Conjugated biological molecules, pharmaceutical compositions and methods

ABSTRACT

Antibody drug conjugates (ADC&#39;s) comprising a drug conjugated to antibody or antigen binding fragments thereof that bind to Globo series antigen disclosed herein, as well as methods of use thereof. Methods of use include, without limitation, cancer therapies and diagnostics. The antibodies of the disclosure can bind to certain cancer cell surfaces. Exemplary targets of the antibodies disclosed herein can include carcinomas, such as sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testical cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer and prostate cancer.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 15/820,309, filed Nov. 21, 2017, which claims the benefit of and priority to U.S. Provisional Patent Application No. 62/424,851, filed on Nov. 21, 2016, both of which are incorporated herein by reference in their entirety.

FIELD

The present disclosure is directed to antibody-drug conjugates (ADCs) compositions and methods of use thereof to treat cancer. Also described herein are methods of using antibody-drug conjugate compounds for treatment of mammalian cells associated with pathological conditions. The present disclosure relates to antibodies and binding fragments thereof to Globo series antigens (Globo H, SSEA-3 and SSEA-4), including pharmaceutical compositions comprising said antibody and/or binding fragments. Further, methods are provided for administering ADCs to a subject in an amount effective to inhibit cancer cells.

BACKGROUND OF THE INVENTION

Numerous surface carbohydrates are expressed in malignant tumor cells. For example, the carbohydrate antigen Globo H (Fuc α 1→2 Gal β 1→3 GalNAc β 1→3 Gal α 1→4 Gal β 14 Glc) was first isolated as a ceramide-linked Glycolipid and identified in 1984 from breast cancer MCF-7 cells. (Bremer E G, et al. (1984) J Biol Chem 259:14773-14777). Previous studies have also shown that Globo H and stage-specific embryonic antigen 3 (Gal β 1→3GalNAc β 1→3Gal α 1→4Gal β 1→4Glc β 1) (SSEA-3, also called Gb5) were observed on breast cancer cells and breast cancer stem cells (WW Chang et al. (2008) Proc Natl Acad Sci USA, 105(33): 11667-11672). In addition, SSEA-4 (stage-specific embryonic antigen-4) (Neu5Ac α 2→3Gal β 1→3GalNAc β 1→3Gal α 1→4Gal β 1→4Glc β 1) has been commonly used as a cell surface marker for pluripotent human embryonic stem cells and has been used to isolate mesenchymal stem cells and enrich neural progenitor cells (Kannagi R et al. (1983) EMBO J, 2:2355-2361). These findings support that Globo series antigens (Globo H, SSEA-3 and SSEA-4) are unique targets for cancer therapies and can be used to direct therapeutic agents to targeting cancer cells effectively. It is of great interest to identify glycan markers associated with and/or predictive of cancers, and develop antibody-drug conjugates (ADCs) against the markers for use in diagnosing and treating a broad spectrum of cancers. Globo series antigens can be designed as an ADC by combining its specific antibodies with therapeutic agents through different linkers.

The use of antibody-drug conjugates (ADCs) for the local delivery of cytotoxic or cytostatic agents, e.g., drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg. Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) theoretically allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, while systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., 1986, Lancet pp. (Mar. 15, 1986):603-05; Thorpe, 1985, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506). Maximal efficacy with minimal toxicity is sought thereby. Both polyclonal antibodies and monoclonal antibodies have been reported as useful in these strategies (Rowland et al., 1986, Cancer Immunol. Immunother. 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., 1986, supra). Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.

The auristatin peptides, auristain E (AE) and monomethylauristatin (MMAE), synthetic analogs of dolastatin, were conjugated to: (i) chimeric monoclonal antibodies cBR96 (specific to Lewis Y on carcinomas); (ii) cAC10 which is specific to CD30 on hematological malignancies (Klussman, et al. (2004), Bioconjugate Chemistry 15(4):765-773; Doronina et al. (2003) Nature Biotechnology 21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation to Ligands”; Francisco et al. (2003) Blood 102(4):1458-1465; U.S. Publication 2004/0018194; (iii) anti-CD20 antibodies such as RITUXAN® (WO 04/032828) for the treatment of CD20-expressing cancers and immune disorders; (iv) anti-EphB2 antibodies 2H9 and anti-IL-8 for treatment of colorectal cancer (Mao, et al. (2004) Cancer Research 64(3):781-788); (v) E-selectin antibody (Bhaskar et al. (2003) Cancer Res. 63:6387-6394); and (vi) other anti-CD30 antibodies (WO 03/043583).

SUMMARY OF THE INVENTION

Accordingly, the present disclosure is based on the discovery that Globo series antigens are aberrantly expressed in a broad spectrum of cancers, but not on normal cells. Cancers expressing Globo series antigens include, but are not limited to, sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testical cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer and prostate cancer.

In one aspect, the present disclosure features an antibody or binding fragment thereof specific to Globo series antigens.

In certain embodiments, the antibody is an Anti-Globo H antibody.

In certain embodiments, the Anti-Globo H antibody is OBI-888. Exemplary OBI antibody 888 is as described in US2017/0101462 (WO2017/062792), the contents of which are incorporated by reference in its entirety.

In certain embodiments, the antibody is an Anti-SSEA4 antibody.

In certain embodiments, the Anti-SSEA4 antibody is OBI-898. Exemplary OBI antibody 898 is as described in US 2017/283488 (WO2017/172990), the contents of which are incorporated by reference in its entirety.

In one aspect, the invention provides antibody-drug conjugates (ADCs), comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In certain embodiments, the present disclosure features an antibody-drug conjugate (ADC) thereof specific to Globo series antigens.

In certain embodiments, the drug is monomethyl auristatin E (MMAE).

In one aspect, the present disclosure provides a method for inhibiting the proliferation of cancer cells, comprising the administering of an effective amount of an exemplary ADC (OBI-999) to a subject in need thereof, wherein the proliferation of cancer cells is inhibited.

In certain embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of the exemplary ADC (OBI-999) described herein.

In the disclosed compositions, both the ADC or any other relevant components are present in immunogenically effective amounts. For each specific ADC, the optimal immunogenically effective amount should be determined experimentally (taking into consideration specific characteristics of a given patient and/or type of treatment). Generally, this amount is in the range of 0.01 μg-250 mg per kilogram body weight of an antibody which was specifically targeting a Globo series antigen. In some embodiments, a therapeutically effective amount of a therapeutic composition (i.e., an effective dosage) may range from about 0.001 μg/kg to about 250 mg/kg, 0.01 μg/kg to 100 mg/kg, or 0.1 μg/kg to 50 mg/kg or about or at least: 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009; 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09; 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, or 250 grams or micrograms per kilogram of patient body weight, or any range between any of the numbers listed herein, or other ranges that would be apparent and understood by artisans without undue experimentation. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present.

In certain embodiments, the cancer is selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testical cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer and prostate cancer.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appending claims.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

A more complete understanding of the invention may be obtained by reference to the accompanying drawings, when considered in conjunction with the subsequent detailed description. The embodiments illustrated in the drawings are intended only to exemplify the invention and should not be construed as limiting the invention to the illustrated embodiments.

FIGS. 1A and 1B show hydrophobic interaction chromatogram (HIC) for OBI-888 (FIG. 1A) and ADC (OBI-999) (FIG. 1B).

FIGS. 2A and 2B show size exclusion chromatogram (SEC) for OBI-888 (FIG. 2A) and ADC (OBI-999) (FIG. 2B).

FIG. 3 showed SDS-PAGE analysis of OBI-888 and ADC (OBI-999). Lane M: Novex Sharp Marker; Lane 1: Native OBI-888 in formulation buffer; Lane 2: Native OBI-888 in reaction buffer; Lane 3: ADC (OBI-999).

FIGS. 4A and 4B show tumor growth curves in MCF-7 implanted female nude (nu/nu) mice. Test substances were administered as 10 mg/kg once weekly×2 weeks (FIG. 4A) and lower doses at 3, 1, and 0.3 mg/kg once weekly×6 weeks (FIG. 4B). T/C value 42% was considered significant anti-tumor activity (#) compared to the vehicle group. Two-way ANOVA followed by Bonferroni post-tests were applied for comparison between the vehicle and test substance-treated groups. Differences are considered significant at *p<0.05.

FIGS. 5A and 5B show body weight changes in MCF-7 implanted female nude (nu/nu) mice. Test substances were administered as 10 mg/kg once weekly×2 weeks (FIG. 5A) and lower doses at 3, 1, and 0.3 mg/kg once weekly×6 weeks (FIG. 5B). T/C value 42% was considered significant anti-tumor activity (#) compared to the vehicle group. Two-way ANOVA followed by Bonferroni post-tests were applied for comparison between the vehicle and test substance-treated groups. Differences are considered significant at *p<0.05.

FIG. 6 showed pictures of female (nu/nu) nude mice with MCF-7 implanted tumors after treatment with Vehicle (25 mM Sodium Citrate, pH 6.5+100 mM NaCl) 10 mL/kg, IV, once weekly×6 weeks.

FIG. 7 showed pictures of female (nu/nu) nude mice with MCF-7 implanted tumors after treatment with Vehicle (25 mM Sodium Citrate, pH 6.5+100 mM NaCl) 10 mL/kg, IV, once weekly×2 weeks.

FIG. 8 showed pictures of female (nu/nu) nude mice with MCF-7 implanted tumors after treatment with ADC (OBI-999) 10 mg/kg, IV, once weekly×2 weeks.

FIG. 9 showed pictures of female (nu/nu) nude mice with MCF-7 implanted tumors after treatment with ADC (OBI-999) 0.3 mg/kg, IV, once weekly×6 weeks.

FIG. 10 showed pictures of female (nu/nu) nude mice with MCF-7 implanted tumors after treatment with ADC (OBI-999) 1 mg/kg, IV, once weekly×6 weeks.

FIG. 11 showed pictures of female (nu/nu) nude mice with MCF-7 implanted tumors after treatment with ADC (OBI-999) 3 mg/kg, IV, once weekly×6 weeks

FIG. 12 showed pictures of female (nu/nu) nude mice with MCF-7 implanted tumors after treatment with OBI-888 10 mg/kg, IV, once weekly×2 weeks.

FIG. 13 showed pictures of female (nu/nu) nude mice with MCF-7 implanted tumors after treatment with OBI-888 0.3 mg/kg, IV, once weekly×6 weeks.

FIG. 14 showed pictures of female (nu/nu) nude mice with MCF-7 implanted tumors after treatment with OBI-888 1 mg/kg, IV, once weekly×6 weeks.

FIG. 15 showed pictures of female (nu/nu) nude mice with MCF-7 implanted tumors after treatment with OBI-888 3 mg/kg, IV, once weekly×6 weeks.

FIG. 16 showed pictures of female (nu/nu) nude mice with MCF-7 implanted tumors after treatment with MMAE 0.057 mg/kg, IV, once weekly×2 weeks.

FIG. 17 showed tumor growth curves in NCI-N87 implanted female nude (nu/nu) mice. Vehicle and test substances were administered as detailed in the study design. T/C value ≤42% was considered significant anti-tumor activity (#) compared to the vehicle group. Two-way ANOVA followed by Bonferroni post-tests were applied for comparison between the vehicle and test substance-treated groups. Differences are considered significant at *p<0.05.

FIG. 18 showed body weight changes in NCI-N87 implanted female nude (nu/nu) mice. Vehicle and test substances were administered as detailed in the study design. The body weights were measured and recorded twice weekly until Day 100.

FIG. 19 showed pictures of female (nu/nu) nude mice with NCI-N87 implanted tumors after treatment with Vehicle (25 mM Sodium Citrate, pH 6.5+100 mM NaCl) 10 mL/kg, IV, once weekly×4 weeks+Vehicle (PBS, pH 7.4) 10 mL/kg, IP, once weekly×4 weeks.

FIG. 20 showed pictures of female (nu/nu) nude mice with NCI-N87 implanted tumors after treatment with ADC (OBI-999) 1 mg/kg, IV, once weekly×4 weeks.

FIG. 21 showed pictures of female (nu/nu) nude mice with NCI-N87 implanted tumors after treatment with ADC (OBI-999) 3 mg/kg, IV, once weekly×4 weeks.

FIG. 22 showed pictures of female (nu/nu) nude mice with NCI-N87 implanted tumors after treatment with ADC (OBI-999) 10 mg/kg, IV, once weekly×4 weeks.

FIG. 23 showed pictures of female (nu/nu) nude mice with NCI-N87 implanted tumors after treatment with OBI-888 10 mg/kg, IV, once weekly×4 weeks.

FIG. 24 showed pictures of female (nu/nu) nude mice with NCI-N87 implanted tumors after treatment with OBI-910 (Anti-CD30 ADC) 3 mg/kg, IV, once weekly×4 weeks.

FIG. 25 showed pictures of female (nu/nu) nude mice with NCI-N87 implanted tumors after treatment with MMAE 0.191 mg/kg, IP, once weekly×4 weeks+OBI-888 10 mg/kg, IV, once weekly×4 weeks.

FIG. 26 showed pictures of female (nu/nu) nude mice with NCI-N87 implanted tumors after treatment with MMAE 0.191 mg/kg, IP, once weekly×4 weeks.

FIG. 27 showed tumor growth curves in NCI-H526 implanted female nude (nu/nu) mice. Vehicle and test substances were administered as detailed in the study design. T/C value ≤42% was considered significant anti-tumor activity (#) compared to the vehicle group. Two-way ANOVA followed by Bonferroni post-tests were applied for comparison between the vehicle and test substance-treated groups. Differences are considered significant at *p<0.05.

FIG. 28 showed body weight changes in NCI-H526 implanted female nude (nu/nu) mice. Vehicle and test substances were administered as detailed in the study design. The body weights were measured and recorded twice weekly until Day 45.

FIG. 29 showed pictures of female (nu/nu) nude mice with NCI-H526 implanted tumors after treatment with Vehicle (25 mM Sodium Citrate, pH 6.5+100 mM NaCl) 10 mL/kg, IV, once weekly×4 weeks+Vehicle (PBS, pH 7.4) 10 mL/kg, IP, once weekly×4 weeks.

FIG. 30 showed pictures of female (nu/nu) nude mice with NCI-H526 implanted tumors after treatment with ADC (OBI-999) 10 mg/kg, IV, once weekly×4 weeks.

FIG. 31 showed pictures of female (nu/nu) nude mice with NCI-H526 implanted tumors after treatment with OBI-888 10 mg/kg, IV, once weekly×4 weeks.

FIG. 32 showed pictures of female (nu/nu) nude mice with NCI-H526 implanted tumors after treatment with MMAE 0.191 mg/kg, IP, once weekly×4 weeks+OBI-888 10 mg/kg, IV, once weekly×4 weeks.

FIG. 33 showed pictures of female (nu/nu) nude mice with NCI-H526 implanted tumors after treatment with MMAE 0.191 mg/kg, IP, once weekly×4 weeks.

FIG. 34 showed tumor growth curves in different treatment groups of male BALB/c nude mice bearing HPAC established tumors. Vehicle and test substances were administered as detailed in the study design. Data points represent group mean, error bars represent standard error of the mean (SEM).

FIG. 35 showed the body weight changes of different treatment groups in male BALB/c nude mice bearing HPAC established tumors. Vehicle and test substances were administered as detailed in the study design. Data points represent group mean body weight. Error bars represent standard error of the mean (SEM).

FIG. 36 showed pictures of different treatment groups in male BALB/c nude mice bearing HPAC established tumors.

FIG. 37 showed the entire chemical structure of OBI-999 (DAR=4).

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, antibody-drug conjugate (ADC) methods and compositions directed to the markers for use in diagnosing and treating a broad spectrum of cancers are provided. An antibody-drug conjugate (ADC) comprising a drug conjugated to an antibody or an antigen-binding fragment that binds Globo series antigens was developed and disclosed herein. Methods of use include, without limitation, cancer therapies and diagnostics. The ADC described herein can bind to a broad spectrum of Globo series antigens-expressing cancer cells, thereby facilitating cancer diagnosis and treatment. Cells that can be targeted by the antibodies include carcinomas, such as those in skin, blood, lymph node, brain, lung, breast, mouse, esophagus, stomach, liver, bile duct, pancreas, colon, kidney, cervix, ovary, prostate cancer, etc. General Definitions

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, by Harlow and Lane s (Cold Spring Harbor Laboratory Press, 1988); and Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986).

As used herein, the term “glycan” refers to a polysaccharide, or oligosaccharide. Glycan is also used herein to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or a proteoglycan. Glycans usually consist solely of 0-glycosidic linkages between monosaccharides. For example, cellulose is a glycan (or more specifically a glucan) composed of β-1,4-linked D-glucose, and chitin is a glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can be homo or heteropolymers of monosaccharide residues, and can be linear or branched. Glycans can be found attached to proteins as in glycoproteins and proteoglycans. They are generally found on the exterior surface of cells. 0- and N-linked glycans are very common in eukaryotes but may also be found, although less commonly, in prokaryotes. N-Linked glycans are found attached to the R-group nitrogen (N) of asparagine in the sequon. The sequon is a Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid except praline.

As used herein, the term “antigen” is defined as any substance capable of eliciting an immune response.

As used herein, the term “immunogenicity” refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.

As used herein, the term “epitope” is defined as the parts of an antigen molecule which contact the antigen binding site of an antibody or a T cell receptor.

As used herein, the term “vaccine” refers to a preparation that contains an antigen, consisting of whole disease-causing organisms (killed or weakened) or components of such organisms, such as proteins, peptides, or polysaccharides, that is used to confer immunity against the disease that the organisms cause. Vaccine preparations can be natural, synthetic or derived by recombinant DNA technology.

As used herein, the term “antigen specific” refers to a property of a cell population such that supply of a particular antigen, or a fragment of the antigen, results in specific cell proliferation.

As used herein, the term “specifically binding,” refers to the interaction between binding pairs (e.g., an antibody and an antigen). In various instances, specifically binding can be embodied by an affinity constant of about 10⁻⁶ moles/liter, about 10⁻⁷ moles/liter, or about 10⁻⁸ moles/liter, or less.

The phrase “substantially similar,” “substantially the same”, “equivalent”, or “substantially equivalent”, as used herein, denotes a sufficiently high degree of similarity between two numeric values (for example, one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values, anti-viral effects, etc.). The difference between said two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% as a function of the value for the reference/comparator molecule.

The phrase “substantially reduced,” or “substantially different”, as used herein, denotes a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value for the reference/comparator molecule.

“Binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described in the following.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which generally lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.

The terms “antibody” and “immunoglobulin” are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, monovalent, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein). An antibody can be chimeric, human, humanized and/or affinity matured.

The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of heavy or light chain of the antibody. These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′)2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

“Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody” are used herein interchangeably, to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain the Fc region.

“Antibody fragments” comprise only a portion of an intact antibody, wherein the portion retains at least one, and as many as most or all, of the functions normally associated with that portion when present in an intact antibody. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment, for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half life modulation, ADCC function and complement binding. In one embodiment, an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody. For example, such an antibody fragment may comprise an antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. Such monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones. It should be understood that the selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler et al., Nature, 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display technologies (See, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO98/24893; WO96/34096; WO96/33735; WO91/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio. Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

Antibodies of the present invention also include chimerized or humanized monoclonal antibodies generated from antibodies of the present invention.

The antibodies can be full-length or can comprise a fragment (or fragments) of the antibody having an antigen-binding portion, including, but not limited to, Fab, F(ab′)2, Fab′, F(ab)′, Fv, single chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragment (e.g., Ward et al, Nature, 341:544-546 (1989)), an CDR, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Single chain antibodies produced by joining antibody fragments using recombinant methods, or a synthetic linker, are also encompassed by the present invention. Bird et al. Science, 1988, 242:423-426. Huston et al, Proc. Natl. Acad. Sci. USA, 1988, 85:5879-5883.

The antibodies or antigen-binding portions thereof of the present invention may be monospecific, bi-specific or multispecific.

All antibody isotypes are encompassed by the present invention, including IgG (e.g., IgG₁, IgG₂, IgG₃, IgG₄), IgM, IgA (IgA₁, IgA₂), IgD or IgE (all classes and subclasses are encompassed by the present invention). The antibodies or antigen-binding portions thereof may be mammalian (e.g., mouse, human) antibodies or antigen-binding portions thereof. The light chains of the antibody may be of kappa or lambda type.

Thus, anti-cancer antibodies of the present invention include in combination with a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, of non-murine origin, preferably of human origin, which can be incorporated into an antibody of the present invention.

Antibodies with a variable heavy chain region and a variable light chain region that are at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%>, at least about 88%>, at least about 89%>, at least about 90%>, at least about 91>, at least about 92%>, at least about 93%>, at least about 94%>, at least about 95%), at least about 96%>, at least about 97%>, at least about 98%>, at least about 99%> or about 100% homologous to the variable heavy chain region and variable light chain region of the antibody produced by the reference antibody, and can also bind to Globo series antigens (Globo H, SSEA-3 and SSEA-4). Homology can be present at either the amino acid or nucleotide sequence level.

The antibodies or antigen-binding portions may be peptides. Such peptides can include variants, analogs, orthologs, homologs and derivatives of peptides, that exhibit a biological activity, e.g., binding of a carbohydrate antigen. The peptides may contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), peptides with substituted linkages, as well as other modifications known in the art.

Also within the scope of the invention are antibodies or antigen-binding portions thereof in which specific amino acids have been substituted, deleted or added. In an exemplary embodiment, these alternations do not have a substantial effect on the peptide's biological properties such as binding affinity. In another exemplary embodiment, antibodies may have amino acid substitutions in the framework region, such as to improve binding affinity of the antibody to the antigen. In yet another exemplary embodiment, a selected, small number of acceptor framework residues can be replaced by the corresponding donor amino acids. The donor framework can be a mature or germline human antibody framework sequence or a consensus sequence. Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science, 247: 1306-1310 (1990). Cunningham et al, Science, 244: 1081-1085 (1989). Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994). T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989). Pearson, Methods Mol. Biol. 243:307-31 (1994). Gonnet et al., Science 256: 1443-45 (1992).

The antibody, or antigen-binding portion thereof, can be derivatized or linked to another functional molecule. For example, an antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent interaction, etc.) to one or more other molecular entities, such as another antibody, a detectable agent, a cytotoxic agent, a pharmaceutical agent, a protein or peptide that can mediate association with another molecule (such as a streptavidin core region or a polyhistidine tag), amino acid linkers, signal sequences, immunogenic carriers, or ligands useful in protein purification, such as glutathione-S-transferase, histidine tag, and staphylococcal protein A. One type of derivatized protein is produced by crosslinking two or more proteins (of the same type or of different types). Suitable crosslinkers include those that are heterobifunctional, having two distinct reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, 111. Useful detectable agents with which a protein can be derivatized (or labeled) include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting, exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, and, phycoerythrin. A protein or antibody can also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, beta-galactosidase, acetylcholinesterase, glucose oxidase and the like. A protein can also be derivatized with a prosthetic group (e.g., streptavidin/biotin and avidin/biotin).

Nucleic acids encoding a functionally active variant of the present antibody or antigen-binding portion thereof are also encompassed by the present invention. These nucleic acid molecules may hybridize with a nucleic acid encoding any of the present antibody or antigen-binding portion thereof under medium stringency, high stringency, or very high stringency conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. 6.3.1-6.3.6, 1989, which is incorporated herein by reference. Specific hybridization conditions referred to herein are as follows: 1) medium stringency hybridization conditions: 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 2) high stringency hybridization conditions: 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and 3) very high stringency hybridization conditions: 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.

A nucleic acid encoding the present antibody or antigen-binding portion thereof may be introduced into an expression vector that can be expressed in a suitable expression system, followed by isolation or purification of the expressed antibody or antigen-binding portion thereof. Optionally, a nucleic acid encoding the present antibody or antigen-binding portion thereof can be translated in a cell-free translation system. U.S. Pat. No. 4,816,567. Queen et al, Proc Natl Acad Sci USA, 86: 10029-10033 (1989).

The present antibodies or antigen-binding portions thereof can be produced by host cells transformed with DNA encoding light and heavy chains (or portions thereof) of a desired antibody. Antibodies can be isolated and purified from these culture supernatants and/or cells using standard techniques. For example, a host cell may be transformed with DNA encoding the light chain, the heavy chain, or both, of an antibody. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding, e.g., the constant region.

The present nucleic acids can be expressed in various suitable cells, including prokaryotic and eukaryotic cells, e.g., bacterial cells, (e.g., E. coli), yeast cells, plant cells, insect cells, and mammalian cells. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC). Non-limiting examples of the cells include all cell lines of mammalian origin or mammalian-like characteristics, including but not limited to, parental cells, derivatives and/or engineered variants of monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2/0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells. The engineered variants include, e.g., glycan profile modified and/or site-specific integration site derivatives.

The present invention also provides for cells comprising the nucleic acids described herein. The cells may be a hybridoma or transfectant.

Alternatively, the present antibody or antigen-binding portion thereof can be synthesized by solid phase procedures well known in the art. Solid Phase Peptide Synthesis: A Practical Approach by E. Atherton and R. C. Sheppard, published by IRL at Oxford University Press (1989). Methods in Molecular Biology, Vol. 35: Peptide Synthesis Protocols (ed. M. W. Pennington and B. M. Dunn), chapter 7. Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984). G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 1 and Vol. 2, Academic Press, New York, (1980), pp. 3-254. M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984).

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and/or capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

The term “hypervariable region”, “HVR”, or “HV”, when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

“Framework” or “FW” residues are those variable domain residues other than the hypervariable region residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO93/1161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

An “affinity matured” antibody is one with one or more alterations in one or more HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In one embodiment, an affinity matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics at least one of the functional activities of a polypeptide of interest.

A “disorder” is any condition that would benefit from treatment with an antibody of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include cancer.

The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

“Tumor” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

As used herein, “treatment” refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing or decreasing inflammation and/or tissue/organ damage, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or disorder.

An “individual” or a “subject” is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice and rats. In certain embodiments, the vertebrate is a human.

“Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. In certain embodiments, the mammal is human.

An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

A “therapeutically effective amount” of a substance/molecule of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.

A “combination” refers to combination therapy would be the amount of the antibody-drug conjugate and/or the amount of other biological or chemical drugs that when administered together (either as co-administration and/or co-formulation), either sequentially or simultaneously, on the same or different days during a treatment cycle, have a synergistic effect that is therapeutically effective and more than therapeutically additive.

The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A “tumor” comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes, (e.g. ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁰C, and radioactive isotopes of lutetium-177, strontium-89 and samarium (153Sm)), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof.

The term “photodynamic therapy (PDT)”, sometimes called photochemotherapy, is a form of phototherapy involving light and a photosensitizing chemical substance, used in conjunction with molecular oxygen to elicit cell death (phototoxicity). It is used clinically to treat a wide range of medical conditions, including wet age-related macular degeneration, psoriasis, atherosclerosis and has shown some efficacy in anti-viral treatments, including herpes. It also treats malignant cancers including head and neck, lung, bladder, skin and prostate cancer (Wang, S S et al. Cancer Journal. 8 (2): 154-63. 2002). The “photodynamic therapeutic agent” is selected from Photofrin, Laserphyrin, Aminolevulinic acid (ALA), Silicon Phthalocyanine Pc 4, m-tetrahydroxyphenylchlorin (mTHPC), chlorin e6 (Ce6), Allumera, Levulan, Foscan, Metvix, Hexvix, Photochlor, Photosens, Photrex, Lumacan, Visonac, Amphinex, Verteporfin, Purlytin, ATMPn, Zinc phthalocyanine (ZnPc), Protoporphyrin IX (PpIX), Pyropheophorbidea (PPa) or Pheophorbide a (PhA).

A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), mertansine (also called DM1), anthracycline, pyrrolobenzodiazepine, α-amanitin, tubulysin, benzodiazepine, erlotinib (TARCEVA®), Genentech/OSI Pharm.), bortezomib (VELCADE®, Millenium Pharm.), fulvestrant (FASLODEX®, Astrazeneca), sunitinib (SUTENT®, SU11248, Pfizer), letrozole (FEMARA®), Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), oxaliplatin (ELOXATIN®, Sanofi), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafarnib (SARASAR®, SCH 66336), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs.), and gefitinib (IRESSA®, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®, Roche); and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Also included in this definition of “chemotherapeutic agent” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON. toremifene; (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVIS OR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (viii) ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME® ribozyme) and a HER2 expression inhibitor; (ix) vaccines such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; (x) anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech); and (xi) pharmaceutically acceptable salts, acids or derivatives of any of the above.

Protein kinase inhibitors include tyrosine kinase inhibitors which inhibit to some extent tyrosine kinase activity of a tyrosine kinase such as an ErbB receptor. Examples of tyrosine kinase inhibitors include EGFR-targeted drugs such as: (i) antibodies which bind to EGFR, including MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBITUX®, Imclone) and reshaped human 225 (H225) (WO 96/40210, Imclone Systems Inc.); antibodies that bind type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind EGFR (U.S. Pat. No. 5,891,996); and human antibodies that bind EGFR, such as ABX-EGF (WO 98/50433); (ii) anti-EGFR antibody conjugated with a cyotoxic agent (EP 659439A2); and small molecules that bind to EGFR including ZD1839 or Gefitinib (IRESSA™; Astra Zeneca), Erlotinib HCl (CP-358774, TARCEVA™; Genentech/OSI) and AG1478, AG1571 (SU 5271; Sugen), quinazolines such as PD 153035,4-(3-chloroanilino) quinazoline, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines, curcumin (diferuloyl methane, 4,5-bis(4-fluoroanilino)phthalimide), tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to ErbB-encoding nucleic acid); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-ErbB inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); Imatinib mesylate (Gleevac; Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxanib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone); or as described in: U.S. Pat. No. 5,804,396; WO 99/09016 (American Cyanamid); WO 98/43960 (American Cyanamid); WO 97/38983 (Warner Lambert); WO 99/06378 (Warner Lambert); WO 99/06396 (Warner Lambert); WO 96/30347 (Pfizer, Inc); WO 96/33978 (Zeneca); WO 96/3397 (Zeneca); and WO 96/33980 (Zeneca).

An “anti-angiogenic agent” refers to a compound which blocks, or interferes with to some degree, the development of blood vessels. The anti-angiogenic factor may, for instance, be a small molecule or antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. An exemplary anti-angiogenic agent is an antibody that binds to Vascular Endothelial Growth Factor (VEGF) such as bevacizumab (AVASTIN®, Genentech).

The term “cytokine” is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

The term “prodrug” as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). The prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified pro drugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.

A “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as the anti-ErbB2 antibodies disclosed herein and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.

The phrase “pharmaceutically acceptable salt,” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of an ADC. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion.

“Pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and an ADC. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

General Features of Exemplary Antibody-Drug Conjugates

The compounds of the invention include those with utility for anticancer activity. In particular, the compounds include an antibody conjugated, i.e. covalently attached by a linker, to a drug moiety where the drug when not conjugated to an antibody has a cytotoxic or cytostatic effect. The biological activity of the drug moiety is thus modulated by conjugation to an antibody. The antibody-drug conjugates (ADCs) of the invention may selectively deliver an effective dose of a cytotoxic agent to tumor tissue whereby greater selectivity, i.e. a lower efficacious dose may be achieved.

Antibody-drug conjugates (ADCs) may be represented by Formula I:

Ab-(L-D)_(n)  (I)

or a pharmaceutically acceptable salt or solvate thereof, wherein: Ab is an antibody which binds Globo series antigen, or which binds to one or more tumor-associated antigens or cell-surface receptors; n is the Drug-to-antibody ratio (DAR) and ranging from 1 to 8.

An antibody-drug conjugate (ADC) comprise an antibody covalently attached by a linker to one or more MMAE moieties. ADC may be represented by Formula I:

Ab-(L-D)_(n)  (I)

wherein one or more MMAE drug moieties (D) are covalently linked by L to an antibody (Ab). Ab is an antibody which targets Globo series antigens or which binds to one or more tumor-associated antigens or cell-surface receptors. The linker L may be stable outside a cell, i.e. extracellular.

In one embodiment, a substantial amount of the drug moiety is not cleaved from the antibody until the antibody-drug conjugate enters a cell with a cell-surface receptor specific for the antibody of the antibody-drug conjugate, and the drug moiety is cleaved from the antibody when the antibody-drug conjugate does enter the cell.

In another embodiment, the ADC specifically binds to a Globo series antigen, such as Globo H, SSEA-3, or SSEA-4. The ADC may specifically bind to Globo H, SSEA-4, SSEA-3. The ADC may inhibit growth of tumor cells which expresses Globo series antigens.

In another embodiment, the antibody (Ab) of Formula I is a human, chimeric or humanized antibody.

Another aspect of the invention is a pharmaceutical composition including a Formula I compound, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable diluent, carrier, or excipient.

Another aspect provides a pharmaceutical combination comprising a Formula I compound and a second compound having anti-cancer properties or other therapeutic effects.

Another aspect includes diagnostic and therapeutic uses for the compounds and compositions disclosed herein.

Another aspect is a method for killing or inhibiting the proliferation of tumor cells or cancer cells comprising treating the cells with an amount of an antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, being effective to kill or inhibit the proliferation of the tumor cells or cancer cells.

Another aspect are methods of treating cancer comprising administering to a patient a formulation of a Formula I compound. One method is for the treatment of cancer in a mammal, wherein the cancer is characterized by the expression of the Globo series antigens. The mammal optionally does not respond, or responds poorly, to treatment with an unconjugated Anti-Globo series antigen antibody. The method comprises administering to the mammal a therapeutically effective amount of an antibody-drug conjugate compound.

Another aspect is a method of inhibiting the growth of tumor cells that expresses Globo H, SSEA-4, and/or SSEA-3 comprising administering to a patient an antibody-drug conjugate compound which binds specifically to said growth factor receptor and a chemotherapeutic agent wherein said antibody-drug conjugate and said chemotherapeutic agent are each administered in amounts effective to inhibit growth of tumor cells in the patient.

Another aspect is a method for the treatment of a human patient susceptible to or diagnosed with a disorder characterized by expression of Globo series antigens, comprising administering a combination of an antibody-drug conjugate compound of Formula I and a chemotherapeutic agent.

Another aspect is an assay method for detecting cancer cells comprising: exposing cells to an antibody-drug conjugate compound, and determining the extent of binding of the antibody-drug conjugate compound to the cells.

Another aspect concerns methods of screening ADC drug candidates for the treatment of a disease or disorder where the disease or disorder is characterized by the expression of Globo series antigens.

Another aspect includes articles of manufacture, i.e. kits, comprising an antibody-drug conjugate, a container, and a package insert or label indicating a treatment.

Another aspect includes methods of treating a disease or disorder characterized by the overexpression of Globo series antigens in a patient with the antibody-drug conjugate compounds.

Another aspect includes methods of making, methods of preparing, methods of synthesis, methods of conjugation, and methods of purification of the antibody-drug conjugate compounds, and the intermediates for the preparation, synthesis, and conjugation of the antibody-drug conjugate compounds.

ADCs: Antibodies:

The antibody unit (Ab-) of Formula I includes within its scope any unit of an antibody that binds or reactively associates or complexes with a receptor, antigen or other receptive moiety associated with a given target-cell population. An antibody can be any protein or protein-like molecule that binds to, complexes with, or reacts with a moiety of a cell population sought to be therapeutically or otherwise biologically modified. In one aspect, the antibody unit acts to deliver the maytansinoid drug moiety to the particular target cell population with which the antibody unit reacts. Such antibodies include, but are not limited to, large molecular weight proteins such as, full-length antibodies and antibody fragments.

Antibodies comprising the antibody-drug conjugates of the invention preferably retain the antigen binding capability of their native, wild type counterparts. Thus, antibodies of the invention are capable of binding, preferably specifically, to antigens.

The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. In one aspect, however, the immunoglobulin is of human, murine, or rabbit origin.

For example, the antibodies can be full-length or can comprise a fragment (or fragments) of the antibody having an antigen-binding portion, including, but not limited to, Fab, F(ab′)2, Fab′, F(ab)′, Fv, single chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragment (e.g., Ward et al., Nature, 341:544-546 (1989)), an isolated CDR, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Single chain antibodies produced by joining antibody fragments using recombinant methods, or a synthetic linker, are also encompassed by the present invention. Bird et al. Science, 1988, 242:423-426. Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85:5879-5883.

For example, the antibodies or antigen-binding portions thereof of the present invention may be monospecific, bi-specific or multispecific. Multispecific or bi-specific antibodies or fragments thereof may be specific for different epitopes of one target carbohydrate (e.g., Globo H) or may contain antigen-binding domains specific for more than one target carbohydrate (e.g., antigen-binding domains specific for Globo H, SSEA-3 and SSEA-4). In one embodiment, a multispecific antibody or antigen-binding portion thereof comprises at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate carbohydrate antigen or to a different epitope on the same carbohydrate antigen. Tutt et al., 1991, J. Immunol. 147:60-69. Kufer et al., 2004, Trends Biotechnol. 22:238-244. The present antibodies can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bi-specific or a multispecific antibody with a second binding specificity.

All antibody isotypes are encompassed by the present invention, including IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD or IgE (all classes and subclasses are encompassed by the present invention). The antibodies or antigen-binding portions thereof may be mammalian (e.g., mouse, human) antibodies or antigen-binding portions thereof. The light chains of the antibody may be of kappa or lambda type.

The variable regions of the present antibodies or antigen-binding portions thereof can be from a non-human or human source. The framework of the present antibodies or antigen-binding portions thereof can be human, humanized, non-human (e.g., a murine framework modified to decrease antigenicity in humans), or a synthetic framework (e.g., a consensus sequence).

In one embodiment, the present antibodies, or antigen-binding portions thereof, comprise at least one heavy chain variable region and/or at least one light chain variable region.

The present antibodies or antigen-binding portions thereof specifically bind to Globo H with a dissociation constant (K_(D)) of less than about 10E-7 M, less than about 10E-8 M, less than about 10E-9 M, less than about 10E-10 M, less than about 10E-11 M, or less than about 10E-12 M. In one embodiment, the antibody or the antibody binding portion thereof has a dissociation constant (K_(D)) of 1^(˜)10×10E-9 or less. In another embodiment, the Kd is determined by surface plasmon resonance.

Antibodies comprising the antibody-drug conjugates of the invention preferably retain the antigen binding capability of their native, wild type counterparts. Thus, antibodies of the invention are capable of binding, preferably specifically, to antigens. Such antigens include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulatory factors, cell proliferation regulatory factors, molecules associated with (for e.g., known or suspected to contribute functionally to) tissue development or differentiation, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in vasculogenesis and molecules associated with (for e.g., known or suspected to contribute functionally to) angiogenesis. The tumor-associated antigen may be a cluster differentiation factor (i.e., a CD protein). An antigen to which an antibody of the invention is capable of binding may be a member of a subset of one of the above-mentioned categories, wherein the other subset(s) of said category comprise other molecules/antigens that have a distinct characteristic (with respect to the antigen of interest).

In one embodiment, the antibody of the antibody-drug conjugates (ADCs) specifically binds to a Globo series antigen Globo H, SSEA-4 and/or SSEA-3.

In some embodiments, the antibodies or antigen-binding portions thereof include, for example, the variable heavy chains and/or variable light chains of the Anti-Globo series antigens antibodies (Globo H: OBI-888, SSEA-4: OBI-999), as shown in Table 1.

In related embodiments, the exemplary antibodies or antigen-binding portions thereof include, for example, the CDRs of the variable heavy chains and/or the CDRs of the variable light chains of Anti-Globo series antigens antibodies (Globo H: OBI-888, SSEA-4: OBI-999). The exemplary CDRs and frameworks of the variable heavy chains and the variable light chains from these hybridoma clones are shown in Table 1.

TABLE 1-1 Anti-Globo H antibody (OBI-888) amino acid sequence [Details described in US2017/0101462 (WO2017/062792)] SEQ Variable Region Amino Acid Sequences ID NO. Heavy Chain CDR1 GFSLYTFDMGVG  1 Heavy Chain CDR2 HIWWDDDKYYNPALKS  2 Heavy Chain CDR3 VRGLHDYYYWFAY  3 Humanized QITLKESGPTLVKPTQTLTLTCTFS  4 Heavy Chain FW1 Humanized WIRQPPGKGLEWLA  5 Heavy Chain FW2 Humanized RLTISKDTSKNQVVLTMTNMDPVDTATYYCAR  6 Heavy Chain FW3 Light Chain CDR1 RASSSVSYMH  7 Light Chain CDR2 ATSNLAS  8 Light Chain CDR3 QQWSRNPFT  9 Humanized EIVLTQSPATLSLSPGERATLSC 10 Light Chain FW1 Humanized WYQQKPGKSPKPWIY 11 Light Chain FW2 Humanized GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC 12 Light Chain FW3 Heavy Chain QITLKESGPTLVKPTQTLTLTCTFSGFSLYTFDMGVGW Variable Region IRQPPGKGLEWLAHIWWDDDKYYNPALKSRLTISKDT 13 of Humanized SKNQVVLTMTNMDPVDTATYYCARVRGLHDYYYWF Antibody AY Light Chain EIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQ 14 Variable Region KPGKSPKPWIYATSNLASGVPSRFSGSGSGTDFTFTISS of Humanized LQPEDIATYYCQQWSRNPFT Antibody Heavy Chain QVTLKESGPGILQPSQTLSLTCSFSGFSLYTFDMGVGW 15 Variable Region of IRQPSGKGLEWLAHIWWDDDKYYNPALKSRLTVSKD Chimeric Antibody TSKNQVFLKIPNVDTADSATYYCARVRGLHDYYYWF AY Light Chain QIVLSQSPTILSASPGEKVTMTCRASSSVSYMHWYQQ 16 Variable Region of KPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISR Chimeric Antibody VEAEDAATYFCQQWSRNPFT Heavy Chain QITLKESGPTLVKPTQTLTLTCTFSGFSLYTFDMGVGW 17 Variable Region of IRQPPGKGLEWLAHIWWDGDKYYNPALKSRLTISKDT Modified Antibody SKNQVVLTMTNMDPVDTATYYCARVRGLHRYYYWF (Humanized R28 AYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA mAb) LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK Light Chain EIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQ 18 Variable Region of KPGKSPKPWIYATSNKASGVPSRFSGSGSGTDFTFTISS Modified Antibody LQPEDIATYYCQQWSRRPFTFGQGTKVEIKRTVAAPS (Humanized R28 VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD mAb) NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC

TABLE 1-2 Anti-SSEA4 antibody (OB1-898) amino acid sequence [Details described in US 2017/283488 (WO2017/172990)] SEQ Variable Region Amino Acid Sequences ID NO. Heavy Chain Variable QVQLKESGPGLVAPSQSLSITCTVSGFSLISYGVDWVR 19 Region (VH) QPPGKGLEWLGVIWGGGNTNYNSSLMSRLSISKDNS KSQVFLKMNSLQTDDTAMYYCAKTGTGYALEYWGQ GTSVTVSS Light Chain Variable ENVLTQSPAIMSASPGEKVTMTCSARSSVSYMHWYQ 20 Region (VL) QKSTASPKLWIYDTSKLASGVPGRFSGSGSGNSYSLTI SSMEAEDVATYYCFQASGYPLTFGAGTKLELKR VL FW1 ENVLTQSPAIMSASPGEKVTMTC 21 VL CDR1 SARSSVSYMH 22 VL FW2 WYQQKSTASPKLWIY 23 VL CDR2 DTSKLAS 24 VL FW3 GVPGRFSGSGSGNSYSLTISSMEAEDVATYYC 25 VL CDR3 FQASGYPLT 26 VL FW4 FGAGTKLELKR 27 VH FW1 QVQLKESGPGLVAPSQSLSITCTVS 28 VH CDR1 GFSLISYGVD 29 VH FW2 WVRQPPGKGLEWLG 30 VH CDR2 VIWGGGNTNYNSSLMS 31 VH FW3 RLSISKDNSKSQVFLKMNSLQTDDTAMYYCAK 32 VH CDR3 TGTGYALEY 33 VH FW4 WGQGTSVTVSS 34

Antibodies with a variable heavy chain region and a variable light chain region that are at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% homologous to the variable heavy chain region and variable light chain region of the antibody produced by clone 2C2, and can also bind to a carbohydrate antigen (e.g. Globo H). Homology can be present at either the amino acid or nucleotide sequence level.

ADC Targeting Globo Series Antigen

One aspect of the present disclosure features the new ADC (OBI-999) specific to Globo H. The Anti-Globo H antibody of the ADC binds to Fucα1→2 Galβ1→3 GalNAcβ1→3 Galα1→4 Galβ1→4 Glc.

Any of the antibodies described herein can be a full length antibody or an antigen-binding fragment thereof. In some examples, the antigen binding fragment is a Fab fragment, a F(ab′)₂ fragment, or a single-chain Fv fragment. In some examples, the antigen binding fragment is a Fab fragment, a F(ab′)₂ fragment, or a single-chain Fv fragment. In some examples, the antibody is a human antibody, a humanized antibody, a chimeric antibody, or a single-chain antibody.

Any of the antibodies described herein has one or more characteristics of: (a) is a recombinant antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, an antibody fragment, a bispecific antibody, a monospecific antibody, a monovalent antibody, an IgG₁ antibody, an IgG₂ antibody, or derivative of an antibody; (b) is a human, murine, humanized, or chimeric antibody, antigen-binding fragment, or derivative of an antibody; (c) is a single-chain antibody fragment, a multibody, a Fab fragment, and/or an immunoglobulin of the IgG, IgM, IgA, IgE, IgD isotypes and/or subclasses thereof; (d) has one or more of the following characteristics: (i) mediates ADCC and/or CDC of cancer cells; (ii) induces and/or promotes apoptosis of cancer cells; (iii) inhibits proliferation of target cells of cancer cells; (iv) induces and/or promotes phagocytosis of cancer cells; and/or (v) induces and/or promotes the release of cytotoxic agents; (e) specifically binds the tumor-associated carbohydrate antigen, which is a tumor-specific carbohydrate antigen; (f) does not bind an antigen expressed on non-cancer cells, non-tumor cells, benign cancer cells and/or benign tumor cells; and/or (g) specifically binds a tumor-associated carbohydrate antigen expressed on cancer stem cells and on normal cancer cells.

Preferably the binding of the antibodies to their respective antigens is specific. The term “specific” is generally used to refer to the situation in which one member of a binding pair will not show any significant binding to molecules other than its specific binding partner (s) and e.g. has less than about 30%, preferably 20%, 10%, or 1% cross-reactivity with any other molecule other than those specified herein.

Production of Antibodies

Various methods have been employed to produce monoclonal antibodies (MAbs). Hybridoma technology, which refers to a cloned cell line that produces a single type of antibody, uses the cells of various species, including mice (murine), hamsters, rats, and humans. Other methods to prepare MAbs, including chimeric and humanized antibodies, uses genetic engineering, i.e. recombinant DNA techniques.

Polyclonal antibodies may be raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, (1984) J. Immunol., 133:3001, and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Binding specificity of monoclonal antibodies produced by hybridoma cells may be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al (1980) Anal. Biochem. 107:220.

DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells (US 2005/0048572; US 2004/0229310). Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al (1993) Curr. Opinion in Immunol. 5:256-262 and Plückthun (1992) Immunol. Revs. 130:151-188.

In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al (1990) Nature 348:552-554; Clackson et al (1991) Nature 352:624-628; and Marks et al (1991) J. Mol. Biol., 222:581-597 describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al (1992) Bio/Technology 10:779-783), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al (1993) Nuc. Acids. Res. 21:2265-2266). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the coding sequence for human heavy chain and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567); and Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81:6851), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.

ADCs: the Linkers: Exemplary ADC Linker

Suitable exemplary linkers for the ADC are described in, for example, U.S. Pat. No. 7,595,292 (WO2005/007197). The entire content directed to linkers is hereby incorporated by reference herein. The linker, L, attaches the antibody to a drug moiety through covalent bond(s), not comprising a disulfide group. The linker is a bifunctional or multifunctional moiety which can be used to link one or more Drug moieties (D) and an antibody unit (Ab) to form antibody-drug conjugates (ADCs) of Formula I. Antibody-drug conjugates (ADCa) can be conveniently prepared using a linker having reactive functionality for binding to the Drug and to the Antibody. A cysteine thiol, or an amine, e.g. N-terminus or amino acid side chain such as lysine, of the antibody (Ab) can form a bond with a functional group of a linker reagent, drug moiety or drug-linker reagent.

The linkers are preferably stable extracellularly. Before transport or delivery into a cell, the antibody-drug conjugate (ADC) is preferably stable and remains intact, i.e. the antibody remains linked to the drug moiety. The linkers are stable outside the target cell and may be cleaved at some efficacious rate inside the cell. An effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow intracellular delivery of the conjugate or drug moiety; (iii) remain stable and intact, i.e. not cleaved, until the conjugate has been delivered or transported to its targeted site; and (iv) maintain a cytotoxic, cell-killing effect or a cytostatic effect of the maytansinoid drug moiety. Stability of the ADC may be measured by standard analytical techniques such as mass spectroscopy, HPLC, and the separation/analysis technique LC/MS.

Covalent attachment of the antibody and the drug moiety requires the linker to have two reactive functional groups, i.e. bivalency in a reactive sense. Bivalent linker reagents which are useful to attach two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups are known, and methods have been described their resulting conjugates (Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, p234-242).

Exemplary ADC Linkers can include biologically active compounds of the general formula II in which one of X and X′ represents a polymer (especially a toxin), and the other represents a hydrogen atom; each Q independently represents a linking group; W represents an electron-withdrawing moiety or a moiety preparable by reduction of an electron-withdrawing moiety; or, if X′ represents a polymer, X-Q-W- together may represent an electron withdrawing group; and in addition, if X represents a polymer, X′ and electron withdrawing group W together with the interjacent atoms may form a ring; each of Z¹ and Z² independently represents a group derived from a biological molecule, each of which is linked to A and B via a nucleophilic moiety; or Z¹ and Z² together represent a single group derived from a biological molecule which is linked to A and B via two nucleophilic moieties; A is a C₁₋₅ alkylene or alkenylene chain; and B is a bond or a C₁₋₄ alkylene or alkenylene chain; are formed by conjugating a suitable polymer to a suitable biologically active molecule via nucleophilic groups in said molecule, preferably via a disulphide bridge.

In certain embodiments, the disclosure provides a protein-polymer conjugate of formula III:

wherein X is a homo- or co-polymer (especially a toxin) selected from the group consisting of polyalkylene glycols, polyvinylpyrrolidones, polyacrylates, polymethacrylates, polyoxazolines, polyvinylalcohols, polyacrylamides, polymethacrylamides, HPMA copolymers, polyesters, polyacetals, poly(ortho ester)s, polycarbonates, poly(imino carbonate)s, polyamides, copolymers of divinylether-maleic anhydride and styrene-maleic anhydride, polysacoharides, and polyglutamic acids; Q is a linking group selected from the group consisting of a direct bond, alkylenes, optionally-substituted aryls, and optionally-substituted heteroaryls, wherein the alkylene, aryl, or heteroaryl may be terminated or interrupted by one or more oxygen atoms, sulphur atoms, keto groups, —O—CO— groups, —CO—O— groups, or —NR groups in which R is an alkyl or aryl group; W is selected from the group consisting of a keto group, an ester group, a sulphone group, a reduced keto group, a reduced ester group, and a reduced sulphone group; X′-Q is hydrogen; A is a C₁₋₅ alkylene or alkenylene chain; B is a bond or a C₁₋₄ alkylene or alkenylene chain; and Z is a single protein linked to A and B via two thiol groups generated by reduction of a disulfide bridge in the protein.

Activity Assays Demonstrating the Efficacy of the Exemplary ADCs

ADC of the invention (OBI-999) can be characterized for their physical/chemical properties and biological functions by various assays known in the art.

Antibodies, or antigen-binding fragments, variants or derivatives thereof of the present disclosure can also be described or specified in terms of their binding affinity to an antigen. The affinity of an antibody for a carbohydrate antigen can be determined experimentally using any suitable method (see, e.g., Berzofsky et al, “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York, N.Y. (1992); and methods described herein). The measured affinity of a particular antibody-carbohydrate antigen interaction can vary if measured under different conditions {e.g., salt concentration, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., K_(D), K_(a), Ka) are preferably made with standardized solutions of antibody and antigen, and a standardized buffer.

The present antibodies or antigen-binding portions thereof have in vitro and in vivo therapeutic, prophylactic, and/or diagnostic utilities. For example, these antibodies can be administered to cells in culture, e.g., in vitro or ex vivo, or to a subject, e.g., in vivo, to treat, inhibit, prevent relapse, and/or diagnose cancer.

Purified antibodies can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion.

Where necessary, antibodies are analyzed for their biological activity. In some embodiments, antibodies of the invention are tested for their antigen binding activity. The antigen binding assays that are known in the art and can be used herein include without limitation any direct or competitive binding assays using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, chemiluminescent immunoassays, nanoparticle immunoassays, aptamer immunoassays, and protein A immunoassays.

Humanized Antibodies

The invention encompasses humanized antibodies. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623.

It is further generally desirable that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

Uses

An ADC of the invention (OBI-999) may be used in, for example, in vitro, ex vivo and in vivo therapeutic methods. ADC of the invention (OBI-999) can be used as an antagonist to partially or fully block the specific antigen activity in vitro, ex vivo and/or in vivo. Accordingly, ADCs of the invention (OBI-999) can be used to inhibit a specific antigen activity, e.g., in a cell culture containing the antigen, in human subjects or in other mammalian subjects having the antigen with which an ADC of the invention (OBI-999) cross-reacts (e.g. chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig or mouse). In one embodiment, an ADC of the invention (OBI-999) can be used for inhibiting antigen activities by contacting the ADC (OBI-999) with the antigen such that antigen activity is inhibited. In one embodiment, the antigen is a human protein molecule.

In one embodiment, an ADC of the invention (OBI-999) can be used in a method for inhibiting an antigen in a subject suffering from a disorder in which the antigen activity is detrimental, comprising administering to the subject an ADC of the invention (OBI-999) such that the antigen activity in the subject is inhibited. In one embodiment, the antigen is a human protein molecule and the subject is a human subject. Alternatively, the subject can be a mammal expressing the antigen with which an ADC of the invention (OBI-999) binds. Still further the subject can be a mammal into which the antigen has been introduced (e.g., by administration of the antigen or by expression of an antigen transgene). An ADC of the invention (OBI-999) can be administered to a human subject for therapeutic purposes. Moreover, an ADC of the invention (OBI-999) can be administered to a non-human mammal expressing an antigen with which the ADC (OBI-999) cross-reacts (e.g., a primate, pig or mouse) for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of ADCs of the invention (OBI-999) (e.g., testing of dosages and time courses of administration). ADCs of the invention (OBI-999) can be used to treat, inhibit, delay progression of, prevent/delay recurrence of, ameliorate, or prevent diseases, disorders or conditions associated with abnormal expression and/or activity of Globo series antigens, including but not limited to cancer, muscular disorders, ubiquitin-pathway-related genetic disorders, immune/inflammatory disorders, neurological disorders, and other ubiquitin pathway-related disorders.

ADCs of the invention (OBI-999) can be used either alone or in combination with other compositions in a therapy. For instance, an ADC of the invention (OBI-999) may be co-administered with another antibody, and/or adjuvant/therapeutic agents (e.g., steroids). For instance, an ADC of the invention (OBI-999) may be combined with an anti-inflammatory and/or antiseptic in a treatment scheme, e.g. in treating any of the diseases described herein, including cancer, muscular disorders, ubiquitin-pathway-related genetic disorders, immune/inflammatory disorders, neurological disorders, and other ubiquitin pathway-related disorders. Such combined therapies noted above include combined administration (where the two or more agents are included in the same or separate formulations), and separate administration, in which case, administration of the ADC of the invention (OBI-999) can occur prior to, and/or following, administration of the adjunct therapy or therapies.

An ADC of the invention (OBI-999) can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the ADC (OBI-999) is suitably administered by pulse infusion, particularly with declining doses of the ADC (OBI-999). Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

Therapeutic Applications

Described herein are therapeutic methods that include administering to a subject in need of such treatment a therapeutically effective amount of a composition that includes one or more ADCs (OBI-999) described herein.

In some embodiments, the subject (e.g., a human patient) in need of the treatment is diagnosed with, suspected of having, or at risk for cancer. Examples of the cancer include, but are not limited to, sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testical cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer or prostate cancer.

In preferred embodiments, the ADC (OBI-999) is capable of targeting Globo series antigens-expressing cancer cells. In some embodiments, the ADC (OBI-999) is capable of targeting Globo series antigens on cancer cells. In some embodiments, the ADC (OBI-999) is capable of targeting Globo series antigens in cancers.

The treatment results in reduction of tumor size, elimination of malignant cells, prevention of metastasis, prevention of relapse, reduction or killing of disseminated cancer, prolongation of survival and/or prolongation of time to tumor cancer progression.

In some embodiments, the treatment further comprises administering an additional therapy to said subject prior to, during or subsequent to said administering of the ADCs (OBI-999). In some embodiments, the additional therapy is treatment with a chemotherapeutic agent. In some embodiments, the additional therapy is radiation therapy.

The methods of the invention are particularly advantageous in treating and preventing early stage tumors, thereby preventing progression to the more advanced stages resulting in a reduction in the morbidity and mortality associated with advanced cancer. The methods of the invention are also advantageous in preventing the recurrence of a tumor or the regrowth of a tumor, for example, a dormant tumor that persists after removal of the primary tumor, or in reducing or preventing the occurrence of a tumor.

In some embodiments, the methods as disclosed herein are useful for the treatment or prevention of a cancer, for example where a cancer is characterized by increased Globo H, SSEA-3 and/or SSEA-4 expression. In some embodiments the cancer comprises a cancer stem cell. In some embodiments, the cancer is a pre-cancer, and/or a malignant cancer and/or a therapy resistant cancer. In some embodiments, the cancer is a brain cancer.

The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subject who needs the treatment may be a human patient having, at risk for, or suspected of having cancer, which include, but not limited to, sarcoma, skin cancer, leukemia, lymphoma, brain cancer, lung cancer, breast cancer, oral cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, pancreas cancer, colon cancer, kidney cancer, cervix cancer, ovary cancer and prostate cancer. A subject having cancer can be identified by routine medical examination.

“An effective amount” as used herein refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who has cancer, a symptom of cancer, or a predisposition toward cancer, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect cancer, the symptom of cancer, or the predisposition toward cancer.

“Development” or “progression” of cancer means initial manifestations and/or ensuing progression of cancer. Development of cancer can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of cancer includes initial onset and/or recurrence.

Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.

Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble ADCs (OBI-999) can be administered by the drip method, whereby a pharmaceutical formulation containing the ADC (OBI-999) and a physiologically acceptable excipients is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients.

Administration of Antibody-Drug Conjugate Pharmaceutical Formulations

Therapeutic antibody-drug conjugates (ADCs) may be administered by any route appropriate to the condition to be treated. The ADC will typically be administered parenterally, i.e. infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal, bolus, intratumor injection or epidural (Shire et al (2004) J. Pharm. Sciences 93(6):1390-1402). Pharmaceutical formulations of therapeutic antibody-drug conjugates (ADCs) are typically prepared for parenteral administration with a pharmaceutically acceptable parenteral vehicle and in a unit dosage injectable form. An antibody-drug conjugate (ADC) having the desired degree of purity is optionally mixed with pharmaceutically acceptable diluents, carriers, excipients or stabilizers, in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.).

Acceptable parenteral vehicles, diluents, carriers, excipients, and stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). For example, lyophilized anti-ErbB2 antibody formulations are described in WO 97/04801, expressly incorporated herein by reference. An exemplary formulation of an ADC such as trastuzumab-SMCC-DM1 contains about 100 mg/ml of trehalose (2-(hydroxymethyl)-6-[3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-3,4,5-triol; C₁₂H₂₂O₁₁; CAS Number 99-20-7) and about 0.1% TWEEN™ 20 (polysorbate 20; dodecanoic acid 2-[2-[3,4-bis(2-hydroxyethoxy)tetrahydrofuran-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl ester; C₂₆H₅₀O₁₀; CAS Number 9005-64-5) at approximately pH 6.

Pharmaceutical formulations of a therapeutic antibody-drug conjugate (ADC) may contain certain amounts of unreacted drug moiety (D), antibody-linker intermediate (Ab-L), and/or drug-linker intermediate (D-L), as a consequence of incomplete purification and separation of excess reagents, impurities, and by-products, in the process of making the ADC; or time/temperature hydrolysis or degradation upon storage of the bulk ADC or formulated ADC composition.

The active pharmaceutical ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi permeable matrices of solid hydrophobic polymers containing the ADC, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

The formulations include those suitable for the foregoing administration routes. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.

The pharmaceutical compositions of ADC may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.

The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 μg of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur. Subcutaneous (bolus) administration may be effected with about 1.5 ml or less of total volume and a concentration of about 100 mg ADC per ml. For ADC that require frequent and chronic administration, the subcutaneous route may be employed, such as by pre-filled syringe or autoinjector device technology.

As a general proposition, the initial pharmaceutically effective amount of ADC administered per dose will be in the range of about 0.01-100 mg/kg, namely about 0.1 to 20 mg/kg of patient body weight per day, with the typical initial range of compound used being 0.3 to 15 mg/kg/day. For example, human patients may be initially dosed at about 1.5 mg ADC per kg patient body weight. The dose may be escalated to the maximally tolerated dose (MTD). The dosing schedule may be about every 3 weeks, but according to diagnosed condition or response, the schedule may be more or less frequent. The dose may be further adjusted during the course of treatment to be at or below MTD which can be safely administered for multiple cycles, such as about 4 or more.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

Although oral administration of protein therapeutics are generally disfavored due to poor bioavailability due to limited absorption, hydrolysis or denaturation in the gut, formulations of ADC suitable for oral administration may be prepared as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the ADC.

The formulations may be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injection immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Exemplary unit dosage formulations contain a daily dose or unit daily sub-dose, or an appropriate fraction thereof, of the active ingredient.

The invention further provides veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier therefore. Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally or by any other desired route.

For the prevention or treatment of disease, the appropriate dosage of an ADC will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. An exemplary dosage of ADC to be administered to a patient is in the range of about 0.1 to about 10 mg/kg of patient weight.

For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. An exemplary dosing regimen comprises administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the anti-ErbB2 antibody. Other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

Combination Therapy

An antibody-drug conjugate (ADC) may be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound having anti-cancer properties. The second compound of the pharmaceutical combination formulation or dosing regimen preferably has complementary activities to the ADC of the combination such that they do not adversely affect each other.

The second compound may be a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, aromatase inhibitor, protein kinase inhibitor, lipid kinase inhibitor, anti-androgen, antisense oligonucleotide, ribozyme, gene therapy vaccine, anti-angiogenic agent and/or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. A pharmaceutical composition containing an ADC may also have a therapeutically effective amount of a chemotherapeutic agent such as a tubulin-forming inhibitor, a topoisomerase inhibitor, or a DNA binder.

Metabolite products may be identified by preparing a radiolabelled (e.g. ¹⁴C or ³H) ADC, administering it parenterally in a detectable dose (e.g. greater than about 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion products from the urine, blood or other biological samples. These products are easily isolated since they are labeled (others are isolated by the use of antibodies capable of binding epitopes surviving in the metabolite). The metabolite structures are determined in conventional fashion, e.g. by MS, LC/MS or NMR analysis. In general, analysis of metabolites is done in the same way as conventional drug metabolism studies well-known to those skilled in the art. The conversion products, so long as they are not otherwise found in vivo, are useful in diagnostic assays for therapeutic dosing of the ADC compounds.

Metabolites include the products of in vivo cleavage of the ADC where cleavage of any bond occurs that links the drug moiety to the antibody. Metabolic cleavage may thus result in the naked antibody, or an antibody fragment. The antibody metabolite may be linked to a part, or all, of the linker. Metabolic cleavage may also result in the production a drug moiety or part thereof. The drug moiety metabolite may be linked to a part, or all, of the linker.

Articles of Manufacture

In another embodiment, an article of manufacture, or “kit”, containing ADC and materials useful for the treatment of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, or blister pack. The containers may be formed from a variety of materials such as glass or plastic. The container holds an antibody-drug conjugate (ADC) composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an ADC. The label or package insert indicates that the composition is used for treating the condition of choice, such as cancer.

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES Example 1: Antibody Drug Conjugation

PolyTherics performed the conjugation of a MMAE reagent to OBI-888 monoclonal antibody to prepare the antibody drug conjugate (ADC; OBI-999). The disulfide conjugation linker is as disclosed in PCT publication number: U.S. Pat. No. 7,595,292 (WO2005/007197); OBI-888 is an Anti-Globo H monoclonal antibody which is as disclosed in US20170101462 (WO2017/062792); monomethyl auristatin E (MMAE) is a commercially available antineoplastic agent. Pilot scale reaction and purification were carried out to identify the appropriate conditions. It was found that the reduced antibody was not prone to aggregation. Subsequent screening of reduction and conjugation conditions resulted in significantly improved conjugation yields. The entire chemical structure of OBI-999 (DAR=4) is indicated in FIG. 37.

Example 2: The Analysis of Antibody Drug Conjugate (OBI-999) 2.1 Appearance

The appearance of the product solution was inspected visually for colour and transparency.

2.2 HIC Analysis

Analytical HIC (hydrophobic interaction chromatography) was carried out using a TOSOH, TSKgel Butyl-NPR column (3.5 cm×4.6 mm) connected to a Dionex Ultimate 3000RS HPLC system. The mobile phase was buffer A (1.5 M ammonium sulfate in 50 mM sodium phosphate, pH 7.0). A gradient was applied using buffer B (20% isopropanol (v/v) in 50 mM sodium phosphate, pH 7.0) from 20% to 86% (over 18.4 min at a flow rate of 1.2 mL/min). The column temperature was maintained at 30° C. throughout the analysis and UV detection was carried out at 280 nm. For each analysis 10 μg of native OBI-888 or conjugated product was injected.

2.3 SEC Analysis

SEC (size exclusion chromatography) was carried out using a TOSOH Bioscience TSKgel Super SW 3000 column (4.6 mm×30 cm, 4 μm) and guard column (4.6 mm×4 cm), connected to an Agilent Infinity 1260 Bioinert system. The mobile phase was 0.2 M Potassium phosphate buffer, pH 6.8 (0.2 M potassium chloride, 15% isopropanol). The flow rate was kept constant at 0.35 mL/min. The column was maintained at ambient temperature throughout the analysis. The analysis was carried out in a 20 min isocratic elution with UV detection at 280 nm. For each analysis, 10 μg of conjugated product was injected. The percentage purity & percentage aggregation present were calculated by comparing the peak areas of the main peaks and early eluting peaks respectively with total peak area.

2.4 SDS-PAGE Analysis

SDS-PAGE analysis was carried out using NuPAGE 4-12% Bis-Tris gels (Invitrogen, Cat #NP0321BOX) under reducing conditions with MES buffer. For analysis, 1 μg of sample (based on protein) was loaded onto the gel per lane. Electrophoresis was carried out at 200 V for 35 min. The gel were stained with InstantBlue (Expedeon, Cat #ISB1LUK) for protein detection and analysed using ImageQuant imaging equipment (GE Healthcare).

2.5 Concentration Determination by Bradford Assay & UV Absorbance.

Concentration of the conjugate was determined against a native OBI-888 standard curve (0-100 μg/mL) by Bradford microplate assay. The assay was performed in a flat bottomed, 96 well plate by mixing 100 μL of each calibration standard and sample with 200 μL of Bradford reagent (Expedeon, BFU1L) in triplicate. The optical density at 595 nm was read and the sample concentration was determined against the native OBI-888 standard curve. The concentration of the conjugate (based on protein) was also determined by UV absorbance (A280) using a Nanodrop spectrophotometer. Measurements were taken in triplicate and the average value was used to determine the antibody concentration:

c=Abs/ε.1 c=concentration (mg/mL); Abs=absorbance at 280 nm; ε=extinction coefficient (mL/mg·cm); l=length (cm).

One ADC sample (OBI-999) was isolated from a larger scale conjugation with a drug to antibody ratio of four and total amount of ADC (OBI-999) isolated was 14.5 mg (by Bradford). The drug to antibody ratio distribution was conducted by using hydrophobic interaction chromatography and showed in FIGS. 1A and 1B. FIG. 1(A) shows a single peak (100%) of OBI-888 by HIC and FIG. 1(B) shows a major peak (82.3%) of ADC (OBI-999) represented the drug to antibody ratio of four. The purities of OBI-888 (FIG. 2A) and ADC (OBI-999) (FIG. 2B) were conducted by using size exclusion chromatography. The purities were both over 96%. Finally, the SDS-PAGE analysis of OBI-888 and ADC (OBI-999) was shown in FIG. 3. The sample was shown to be a homogenous product (>82% single drug to antibody ratio) with low aggregation (<5%). The analytical summary was listed in Table 2.

TABLE 2 The analytical summary of ADC (OBI-999) Analysis Results Appearance Clear colorless solution % Purity (HIC) Drug to Antibody Ratio = 3: 13.4% Drug to Antibody Ratio = 4: 82.3% Drug to Antibody Ratio = >4: 4.3% % Purity (SEC) 96.9% monomeric Amount (by Bradford) 14.5 mg

Example 3: Measurement of the Anti-Tumor Activity of the Exemplary Antibody in Nude Mice (Breast Cancer)

In a xenograft tumor model of human breast adenocarcinoma, MCF-7 (ATCC HTB-22) cells were subcutaneously (SC) implanted (2.0×10⁷ cells in 1:1 matrigel/media mixture at 0.2 mL/mouse) into the right flank of female athymic (nu/nu) nude mice. Supplemental injections of estradiol cyclopentyl propionate (100 μg/mouse) were administered subcutaneously between the scapulae twice weekly, from one week prior to cell implantation to study completion. Tumor-implanted mice were divided into eleven treatment groups, each group containing six animals, and test agent administrations were initiated one day after cell implantation (denoted as Day 1).

3.1 Test Substances and Dosing Pattern

Test substances ADC (OBI-999), OBI-888, and MMAE were formulated by diluting stock with a 25 mM sodium citrate, 100 mM NaCl buffer (pH 6.5) daily and administered intravenously (IV) once weekly for two or six weeks. Two control groups received intravenous injections of vehicle (25 mM Sodium Citrate, pH 6.5+100 mM NaCl) once weekly for either six weeks (group 1) or two weeks (group 2). Test substance, ADC (OBI-999), was dosed at 10 mg/kg once weekly for 2 weeks, and at 0.3, 1, and 3 mg/kg once weekly for six weeks. Test substance, OBI-888, was dosed at 10 mg/kg once weekly for 2 weeks, and at 0.3, 1 and 3 mg/kg once weekly for six weeks. Test substance, MMAE, was dosed at 0.057 mg/kg once weekly for six weeks. All test substances were administered in a dose volume of 10 mL/kg except ADC (OBI-999) was administered at 10 mg/kg with the dose volume of 12.5 mL/kg.

TABLE 3 Study Design for Anti-Tumor Activity of the exemplary antibody in Nude Mice (Breast cancer) Mice Conc. Dosage (nu/nu) Group Test Compound Route mg/mL mL/kg mg/kg (female) 1 Vehicle^(a) IV NA 10    0 × 6^(c) 6 2 Vehicle^(a) IV NA 10    0 × 2^(b) 6 3 ADC (OBI-999) IV 0.8 12.5   10 × 2^(b) 6 4 ADC (OBI-999) IV 0.03 10  0.3 × 6^(c) 6 5 ADC (OBI-999) IV 0.1 10    1 × 6^(c) 6 6 ADC (OBI-999) IV 0.3 10    3 × 6^(c) 6 7 OBI-888 IV 1 10   10 × 2^(b) 6 8 OBI-888 IV 0.03 10  0.3 × 6^(c) 6 9 OBI-888 IV 0.1 10    1 × 6^(c) 6 10 OBI-888 IV 0.3 10    3 × 6^(c) 6 11 MMAE IV 0.0057 10 0.057 × 6^(c) 6 ^(a)25 mM Sodium Citrate, pH 6.5 + 100 mM NaCl ^(b)Dosing: once weekly for 2 weeks starting day after tumor implantation (Day 1) ^(c)Dosing: once weekly for 6 weeks starting day after tumor implantation (Day 1) Monitor and provide tumor size and body weight record twice a week till Day 43 or the tumor size reaches 500 mm³. Tumor photographed at the endpoint of study.

3.2 Cell Line

Human breast adenocarcinoma tumor cell line, MCF-7 (ATCC HTB-22, breast adenocarcinoma), was provided by the Sponsor. The tumor cells were prepared and cultured by the Sponsor (1×10⁸ cells/mL), and 0.2 mL MCF-7 tumor cell inoculum containing 2×10⁷ cells (mixture of matrigel and medium; 1:1) was implanted subcutaneously in the right flank of each mouse.

3.3 Animals

Female (nu/nu) nude mice aged 6-7 weeks obtained from BioLasco Taiwan (under Charles River Laboratories Licensee) were used. The animals were housed in individually ventilated cages (IVC, 36 Mini Isolator System). The allocation for 3-5 animals was 27×20×14 in cm. All animals were maintained in a hygienic environment under controlled temperature (20-24° C.) and humidity (30-70%) with 12-hour light/dark cycle. Free access to standard lab diet (Oriental Yeast Co., Ltd., Japan) and autoclaved tap water were granted. All aspects of this work including housing, experimentation, and animal disposal were performed in general accordance with the “Guide for the Care and Use of Laboratory Animals: Eighth Edition” (National Academies Press, Washington, D.C., 2011) in our AAALAC-accredited laboratory animal facility. In addition, the animal care and use protocol was reviewed and approved by the IACUC at Eurofins Panlabs Taiwan, Ltd.

3.4 Chemicals

Estol-Depot Inj. (estradiol cyclopentyl propionate) (Astar, Taiwan) and BD Matrigel Matrix (BD Biosciences, US) were used in this experiment.

3.5 Equipment

Calipers (Mitutoyo, Japan), Centrifuge 5810R (Eppendorf, Germany), CO₂ Incubator (Forma Scientific Inc., USA), Hemacytometer (Hausser Scientific Horsham, USA), Individually Ventilated Cages (36 Mini Isolator system, Tecniplast, Italy), Inverted Microscope CK-40 (Olympus, Japan), System Microscope E-400 (Nikon, Japan) and Vertical laminar flow (Tsao-Hsin, Taiwan).

3.6 Methods

The tumor volumes, body weights, mortality, and signs of overt toxicity were monitored and recorded twice weekly for 77 days. Tumor volume (mm³) was calculated according to the formula for a prolate ellipsoid: length (mm)×[width (mm)]²×0.5. Tumor growth inhibition was calculated as T/C (treatment/control)×100%. A T/C value ≤42% was considered significant anti-tumor activity. Two-way ANOVA followed by Bonferroni test was used to ascertain the statistical significance of groups compared to respective vehicle control (*p<0.05).

3.7 Results

TABLE 4-1 Tumor volume, Xenograft, Breast, MCF-7 in Nude Mice (Day 1-Day 26) Dose (mg/kg) Tumor Volume (mm³) Gr. Treatment (Route) No. Day1 Day5 Day8 Day12 Day15 Day19 Day22 Day26  1 Vehicle 10 mL/ 1 131 119 133 134 175 220 240 258 (25 mM Sodium kg × 6 2 171 115 160 168 164 219 240 296 Citrate, IV 3 173 137 150 150 176 194 243 286 pH 6.5 + (Once 4 155 125 121 171 142 185 202 240 100 mM weekly) 5 166 117 123 181 138 169 171 203 NaCl) 6 157 125 139 157 171 228 275 306 Mean 159 123 138 160 161 203 229 265 SEM 6 3 6 7 7 10 15 16  2 Vehicle 10 mL/ 1 169 148 137 207 210 268 300 322 (25 mM Sodium kg × 2 2 149 137 146 189 189 234 282 337 Citrate, IV 3 169 139 148 262 279 300 307 317 pH 6.5 + (Once 4 184 133 139 133 123 127 146 231 100 mM weekly) 5 143 113 113 184 131 154 205 210 NaCl) 6 160 121 127 142 153 174 174 166 Mean 162 132 135 186 181 210 236 264 SEM 6 5 5 19 24 28 28 29 % T/C 102 107 98 116 112 103 103 100  3 ADC (OBI-999) 10 mg/ 1 155 126 119 168 104 97 108 89 kg × 2 2 139 123 115 123 90 89 89 94 IV 3 164 117 121 131 89 76 85 74 (Once 4 152 119 110 88 100 97 85 75 weekly) 5 166 110 108 87 94 57 56 54 6 127 125 118 129 101 104 93 85 Mean 151 120 115 121 96 87 86 79 SEM 6 2 2 12 3 7 7 6 % T/C 93 91 85 65 53 41 36 30  4 ADC (OBI-999) 0.3 mg/ 1 139 117 113 159 123 160 160 152 kg × 6 2 176 139 131 143 141 144 176 195 IV 3 146 121 143 155 125 174 187 220 (Once 4 153 119 126 168 156 186 198 197 weekly) 5 148 117 94 146 130 154 155 124 6 135 103 113 141 143 145 163 166 Mean 150 119 120 152 136 161 173 176 SEM 6 5 7 4 5 7 7 14 % T/C 94 97 87 95 84 79 76 66  5 ADC (OBI-999) 1 mg/ 1 197 161 149 175 145 135 138 125 kg × 6 2 162 101 107 74 95 113 110 78 IV 3 157 131 148 126 124 148 135 121 (Once 4 152 133 125 136 124 144 141 120 weekly) 5 131 101 108 127 113 106 117 112 6 116 104 112 108 73 76 67 65 Mean 153 122 125 124 112 120 118 104 SEM 11 10 8 14 10 11 11 10 % T/C 96 99 91 78 70 59 52 39  6 ADC (OBI-999) 3 mg/ 1 156 129 129 117 98 89 93 79 kg × 6 2 194 108 108 125 88 86 88 70 IV 3 129 112 83 72 44 38 24 21 (Once 4 139 108 94 88 81 82 51 37 weekly) 5 143 111 108 80 74 76 55 45 6 139 108 94 88 81 88 83 55 Mean 150 113 103 95 78 77 66 51 SEM 9 3 7 9 8 8 11 9 % T/C 94 92 75 59 48 38 29 19  7 OBI-888 10 mg/ 1 123 94 123 100 162 161 154 137 kg × 2 2 155 114 141 123 161 207 207 214 IV 3 150 97 127 111 104 115 133 145 (Once 4 144 125 123 113 109 106 106 101 weekly) 5 159 125 100 120 145 187 202 213 6 141 110 110 117 108 130 133 125 Mean 145 111 121 114 132 151 156 156 SEM 5 5 6 3 11 17 17 19 % T/C 90 84 90 61 73 74 68 59  8 OBI-888 0.3 mg/ 1 154 110 111 106 119 131 133 98 kg × 6 2 231 123 104 106 111 141 157 197 IV 3 129 104 137 123 117 167 189 203 (Once 4 153 119 117 106 113 113 119 115 weekly) 5 157 98 123 121 108 142 181 180 6 150 101 127 101 104 109 121 164 Mean 162 109 120 111 112 134 150 160 SEM 14 4 5 4 2 9 12 18 % T/C 102 89 87 69 70 66 66 60  9 OBI-888 1 mg/ 1 146 133 113 115 83 97 92 86 kg × 6 2 164 113 127 113 119 146 141 133 IV 3 127 63 69 80 69 81 89 88 (Once 4 146 139 108 129 94 144 122 119 weekly) 5 215 136 115 130 145 200 198 206 6 146 119 106 109 93 119 119 122 Mean 157 117 106 113 101 131 127 126 SEM 12 12 8 7 11 17 16 18 % T/C 99 95 77 71 63 65 55 48 10 OBI-888 3 mg/ 1 146 108 127 87 88 96 92 115 kg × 6 2 137 125 131 125 115 124 137 153 IV 3 126 94 109 94 93 95 99 119 (Once 4 136 119 125 124 124 143 138 114 weekly) 5 135 84 89 91 69 85 86 77 6 181 108 129 121 91 103 108 102 Mean 144 106 118 107 97 108 110 113 SEM 8 6 7 7 8 9 9 10 % T/C 91 86 86 67 60 53 48 43 11 MMAE 0.057 mg/ 1 162 145 139 133 127 125 137 119 kg × 6 2 186 104 131 115 105 121 138 154 IV 3 152 106 131 103 137 148 164 179 (Once 4 188 128 146 129 121 135 143 144 weekly) 5 141 110 121 101 102 137 123 135 6 139 123 125 104 101 113 137 127 Mean 161 119 132 114 116 130 140 143 SEM 9 6 4 6 6 5 5 9 % T/C 101 97 96 71 72 64 61 54

TABLE 4-2 Tumor volume, Xenograft, Breast, MCF-7 in Nude Mice (Day 29-Day 49) Dose (mg/kg) Tumor Volume (mm³) Gr. Treatment (Route) No. Day29 Day33 Day36 Day40 Day43 Day46 Day49  1 Vehicle 10 mL/ 1 281 312 343 372 399 435 455 (25 mM Sodium kg × 6 2 295 325 340 348 368 376 419 Citrate, IV 3 307 328 351 363 388 432 465 pH 6.5 + (Once 4 255 277 295 307 330 355 387 100 mM weekly) 5 214 228 243 259 307 321 351 NaCl) 6 316 370 386 424 432 436 476 Mean 278 307 326 346 371 393 426 SEM 16 20 20 23 19 20 20  2 Vehicle 10 mL/ 1 356 389 432 458 503 612 738 (25 mM Sodium kg × 2 2 344 364 402 402 429 470 484 Citrate, IV 3 381 402 411 415 415 433 456 pH 6.5 + (Once 4 252 279 341 389 422 451 499 100 mM weekly) 5 266 317 325 332 340 356 368 NaCl) 6 169 189 194 197 203 203 203 Mean 295 323 351 366 385 421 458 SEM 33 33 36 38 42 55 72 % T/C 106 105 108 106 104 107 108  3 ADC (OBI-999) 10 mg/ 1 73 66 66 57 53 51 49 kg × 2 2 91 85 83 70 66 53 48 IV 3 75 79 79 62 56 49 45 (Once 4 75 76 78 72 65 62 56 weekly) 5 48 42 39 32 32 31 30 6 86 82 79 66 62 60 58 Mean 75 72 71 60 56 51 48 SEM 6 7 7 6 5 5 4 % T/C 27 23 22 17 15 13 11  4 ADC (OBI-999) 0.3 mg/ 1 176 222 240 269 296 307 340 kg × 6 2 218 238 260 270 276 295 333 IV 3 284 388 405 580 700 756 828 (Once 4 241 254 264 285 312 326 353 weekly) 5 143 168 190 198 209 221 233 6 174 211 225 234 243 259 269 Mean 206 247 264 306 339 361 393 SEM 21 31 30 56 74 81 89 % T/C 74 80 81 88 91 92 92  5 ADC (OBI-999) 1 mg/ 1 140 140 140 153 165 167 182 kg × 6 2 85 95 117 127 147 154 158 IV 3 141 160 174 181 192 179 187 (Once 4 126 143 143 154 180 200 219 weekly) 5 121 113 121 127 127 129 137 6 69 56 64 70 72 74 74 Mean 114 118 127 135 147 151 160 SEM 12 16 15 15 18 18 21 % T/C 41 38 39 39 40 38 38  6 ADC (OBI-999) 3 mg/ 1 79 79 73 60 58 56 56 kg × 6 2 86 83 80 56 53 51 50 IV 3 18 0 0 0 0 0 0 (Once 4 37 40 47 31 31 29 0 weekly) 5 39 44 51 30 29 25 23 6 59 41 39 0 0 0 0 Mean 53 48 48 30 29 27 22 SEM 11 12 12 11 10 10 11 % T/C 19 16 15 9 8 7 5  7 OBI-888 10 mg/ 1 174 200 249 272 292 313 332 kg × 2 2 236 244 273 291 296 293 308 IV 3 139 173 202 249 292 354 425 (Once 4 117 111 119 117 122 128 134 weekly) 5 241 247 264 296 312 347 378 6 159 174 197 200 205 213 230 Mean 178 192 217 238 253 275 301 SEM 21 21 24 28 30 36 43 % T/C 64 63 67 69 68 70 71  8 OBI-888 0.3 mg/ 1 97 110 119 127 137 167 179 kg × 6 2 213 265 331 385 416 486 508 IV 3 211 217 240 246 265 286 310 (Once 4 106 104 121 139 150 152 152 weekly) 5 241 294 372 397 455 493 539 6 189 217 245 274 287 295 298 Mean 176 201 238 261 285 313 331 SEM 25 32 43 47 54 61 66 % T/C 63 65 73 75 77 80 78  9 OBI-888 1 mg/ 1 104 108 119 117 127 133 139 kg × 6 2 153 160 174 181 187 192 200 IV 3 98 127 136 158 178 199 211 (Once 4 123 139 145 147 161 163 178 weekly) 5 255 331 354 379 416 450 491 6 142 145 157 168 185 199 201 Mean 146 168 181 192 209 223 237 SEM 23 33 35 39 42 47 52 % T/C 53 55 56 55 56 57 56 10 OBI-888 3 mg/ 1 123 139 146 152 162 181 192 kg × 6 2 167 181 231 256 269 272 292 IV 3 149 171 183 207 221 236 248 (Once 4 117 145 154 163 166 170 174 weekly) 5 79 83 86 91 101 112 116 6 103 107 107 105 110 112 116 Mean 123 138 151 162 172 181 190 SEM 13 15 21 25 26 26 29 % T/C 44 45 46 47 46 46 45 11 MMAE 0.057 mg/ 1 119 133 152 156 186 222 235 kg × 6 2 156 168 201 223 242 258 301 IV 3 194 216 256 285 296 332 351 (Once 4 160 177 218 226 240 259 264 weekly) 5 146 162 171 184 186 204 210 6 131 152 154 186 239 261 270 Mean 151 168 192 210 232 256 272 SEM 11 11 17 18 17 18 20 % T/C 54 55 59 61 63 65 64

TABLE 4-3 Tumor volume, Xenograft, Breast, MCF-7 in Nude Mice (Day 53-Day 77) Dose (mg/kg) Tumor Volume (mm³) Gr. Treatment (Route) No. Day53 Day56 Day60 Day63 Day67 Day70 Day74 Day77  1 Vehicle 10 mL/ 1 489 519 535 565 645 684 744 853 (25 mM Sodium kg × 6 2 445 469 509 519 557 579 584 601 Citrate, IV 3 519 578 688 785 890 972 986 1155 pH 6.5 + (Once 4 405 416 454 465 514 518 578 796 100 mM weekly) 5 375 450 509 579 622 652 681 881 NaCl) 6 499 530 585 629 752 776 862 1032 Mean 455 494 547 590 663 697 739 886 SEM 23 24 33 45 56 66 66 78  2 Vehicle 10 mL/ 1 803 950 1113 1247 1439 1509 1870 2222 (25 mM Sodium kg × 2 2 488 528 547 575 597 627 663 780 Citrate, IV 3 465 494 515 525 578 583 647 828 pH 6.5 + (Once 4 605 708 793 877 968 1014 1030 1102 100 mM weekly) 5 368 407 414 423 465 465 535 754 NaCl) 6 208 222 243 267 361 433 526 615 Mean 490 552 604 652 735 772 879 1050 SEM 83 103 125 145 164 170 212 243 % T/C 108 112 110 111 111 111 119 119  3 ADC (OBI-999) 10 mg/ 1 48 47 46 45 44 42 42 40 kg × 2 2 46 45 44 42 41 41 41 43 IV 3 45 43 41 39 37 37 37 37 (Once 4 55 56 55 48 46 45 44 43 weekly) 5 29 55 55 23 23 23 23 25 6 58 29 29 55 53 51 51 50 Mean 47 46 45 42 41 40 40 40 SEM 4 4 4 4 4 4 4 3 % T/C 10 9 8 7 6 6 5 5  4 ADC (OBI-999) 0.3 mg/ 1 356 385 411 428 490 607 665 834 kg × 6 2 361 371 444 476 536 630 681 819 IV 3 992 1120 1276 1299 1532 1882 1950 2177 (Once 4 400 484 530 575 641 725 849 1028 weekly) 5 235 241 276 298 362 408 473 575 6 272 292 328 371 390 492 545 704 Mean 436 482 544 575 659 791 861 1023 SEM 114 132 151 150 180 223 224 239 % T/C 96 98 99 97 99 113 117 115  5 ADC (OBI-999) 1 mg/ 1 188 197 205 222 237 264 292 373 kg × 6 2 160 176 183 205 217 245 256 277 IV 3 197 200 219 228 228 233 233 231 (Once 4 236 257 296 331 409 497 552 770 weekly) 5 145 172 195 224 259 291 304 368 6 70 73 74 76 77 77 78 78 Mean 166 179 195 214 238 268 286 350 SEM 23 25 29 33 43 55 63 95 % T/C 36 36 36 36 36 38 39 40  6 ADC (OBI-999) 3 mg/ 1 54 52 52 51 51 51 51 52 kg × 6 2 49 47 46 44 42 42 42 41 IV 3 0 0 0 0 0 0 0 0 (Once 4 0 0 0 0 0 0 0 0 weekly) 5 23 22 21 21 21 21 21 23 6 0 0 0 0 0 0 0 0 Mean 21 20 20 19 19 19 19 19 SEM 10 10 10 10 9 9 9 9 % T/C 5 4 4 3 3 3 3 2  7 OBI-888 10 mg/ 1 377 399 420 496 940 1180 1250 1710 kg × 2 2 333 348 372 436 451 died died died IV 3 610 162 188 701 746 849 952 1476 (Once 4 160 462 519 194 198 265 313 402 weekly) 5 441 239 242 547 605 609 677 768 6 233 657 682 262 268 271 284 325 Mean 359 378 404 439 535 635 695 936 SEM 65 71 74 76 116 175 186 281 % T/C 79 77 74 74 81 91 94 106  8 OBI-888 0.3 mg/ 1 181 202 213 243 284 350 419 658 kg × 6 2 528 573 711 717 744 781 955 1080 IV 3 332 155 157 428 451 510 552 589 (Once 4 152 733 841 157 157 157 157 162 weekly) 5 673 318 341 910 992 1138 1180 1392 6 307 372 381 353 378 436 475 616 Mean 362 392 441 468 501 562 623 750 SEM 83 91 112 118 127 142 153 175 % T/C 80 79 81 79 76 81 84 85  9 OBI-888 1 mg/ 1 147 161 163 168 183 194 201 221 kg × 6 2 259 265 322 360 385 429 475 596 IV 3 220 226 248 337 365 379 443 579 (Once 4 213 653 784 254 321 335 341 377 weekly) 5 632 210 217 847 883 932 962 1289 6 203 240 273 223 228 291 299 394 Mean 279 293 335 365 394 427 454 576 SEM 72 73 93 101 103 106 109 154 % T/C 61 59 61 62 59 61 61 65 10 OBI-888 3 mg/ 1 203 324 356 303 352 389 460 629 kg × 6 2 309 316 381 356 378 378 396 603 IV 3 294 176 179 411 469 508 581 678 (Once 4 174 125 138 181 181 181 181 215 weekly) 5 124 127 131 143 162 177 206 270 6 121 219 269 134 137 139 159 161 Mean 204 215 242 255 280 295 331 426 SEM 33 36 45 48 56 61 71 96 % T/C 45 44 44 43 42 42 45 48 11 MMAE 0.057 mg/ 1 268 325 383 494 598 603 681 760 kg × 6 2 319 329 373 418 455 493 570 632 IV 3 373 399 453 496 505 523 568 807 (Once 4 270 328 358 412 489 528 695 887 weekly) 5 216 305 312 286 307 333 365 410 6 291 249 262 422 458 591 625 922 Mean 290 323 357 421 469 512 584 736 SEM 22 20 27 31 39 40 49 77 % T/C 64 65 65 71 71 73 79 83

TABLE 5-1 Body weight, Xenograft, Breast, MCF-7 in Nude Mice (Day 1-Day 26) Dose (mg/kg) Body Weight (g) Gr. Treatment (Route) No. Day1 Day5 Day8 Day12 Day15 Day19 Day22 Day26  1 Vehicle 10 mL/ 1 25 25 26 27 27 26 26 25 (25 mM Sodium kg × 6 2 24 24 26 28 29 29 29 27 Citrate, IV 3 23 24 25 26 26 26 26 27 pH 6.5 + (Once 4 23 24 26 27 27 27 26 26 100 mM weekly) 5 23 24 25 27 27 27 28 27 NaCl) 6 21 22 24 24 24 24 25 24 Mean 23.2 23.8 25.3 26.5 26.7 26.5 26.7 26.0 SEM 0.5 0.4 0.3 0.6 0.7 0.7 0.6 0.5  2 Vehicle 10 mL/ 1 21 23 23 25 25 25 25 25 (25 mM Sodium kg × 2 2 20 21 22 24 25 25 25 25 Citrate, IV 3 18 19 20 21 22 22 23 22 pH 6.5 + (Once 4 24 24 24 26 26 25 26 27 100 mM weekly) 5 23 25 27 28 28 27 27 28 NaCl) 6 23 24 25 26 27 26 27 27 Mean 21.5 22.7 23.5 25.0 25.5 25.0 25.5 25.7 SEM 0.9 0.9 1.0 1.0 0.8 0.7 0.6 0.9 P < 0.05  3 ADC (OBI-999) 10 mg/ 1 22 22 25 26 27 26 26 26 kg × 2 2 21 21 22 23 23 23 24 23 IV 3 23 25 26 26 27 27 26 27 (Once 4 21 20 21 21 23 21 22 21 weekly) 5 20 20 21 22 23 23 24 24 6 22 23 23 24 24 24 25 25 Mean 21.5 21.8 23.0 23.7 24.5 24.0 24.5 24.3 SEM 0.4 0.8 0.9 0.8 0.8 0.9 0.6 0.9 P < 0.05  4 ADC (OBI-999) 0.3 mg/ 1 22 22 23 24 25 25 25 25 kg × 6 2 23 24 25 27 26 25 26 26 IV 3 21 21 22 23 23 23 25 25 (Once 4 23 23 24 25 24 25 25 26 weekly) 5 21 22 23 24 24 24 25 24 6 21 21 23 24 24 25 25 25 Mean 21.8 22.2 23.3 24.5 24.3 24.5 25.2 25.2 SEM 0.4 0.5 0.4 0.6 0.4 0.3 0.2 0.3 P < 0.05  5 ADC (OBI-999) 1 mg/ 1 22 22 24 24 24 24 24 24 kg × 6 2 22 23 25 26 26 26 26 26 IV 3 21 20 22 22 23 23 24 23 (Once 4 23 25 25 26 27 26 27 26 weekly) 5 22 23 24 25 25 25 25 25 6 23 24 25 26 27 25 26 25 Mean 22.2 22.8 24.2 24.8 25.3 24.8 25.3 24.8 SEM 0.3 0.7 0.5 0.7 0.7 0.5 0.5 0.5  6 ADC (OBI-999) 3 mg/ 1 22 22 24 26 26 26 26 26 kg × 6 2 22 21 23 24 23 23 23 24 IV 3 22 22 23 24 24 24 24 24 (Once 4 22 23 24 26 26 26 26 26 weekly) 5 22 22 22 23 23 23 23 23 6 20 21 22 23 24 24 24 23 Mean 21.7 21.8 23.0 24.3 24.3 24.3 24.3 24.3 SEM 0.3 0.3 0.4 0.6 0.6 0.6 0.6 0.6 P < 0.05  7 OBI-888 10 mg/ 1 21 21 22 23 24 23 24 24 kg × 2 2 24 24 25 26 27 27 28 28 IV 3 21 22 23 24 25 24 24 24 (Once 4 22 24 24 25 26 26 26 26 weekly) 5 21 21 22 23 25 25 25 25 6 21 22 24 23 25 24 25 24 Mean 21.7 22.3 23.3 24.0 25.3 24.8 25.3 25.2 SEM 0.5 0.6 0.5 0.5 0.4 0.6 0.6 0.7 P < 0.05  8 OBI-888 0.3 mg/ 1 21 23 23 25 25 25 25 24 kg × 6 2 19 19 21 22 22 22 23 22 IV 3 19 19 21 22 23 23 24 24 (Once 4 20 21 22 23 23 23 24 23 weekly) 5 21 23 23 23 24 24 24 24 6 21 22 23 22 22 20 19 21 Mean 20.2 21.2 22.2 22.8 23.2 22.8 23.2 23.0 SEM 0.4 0.7 0.4 0.5 0.5 0.7 0.9 0.5 P < 0.05  9 OBI-888 1 mg/ 1 20 21 23 24 25 25 25 25 kg × 6 2 20 20 21 22 23 22 23 23 IV 3 18 19 20 21 21 21 22 21 (Once 4 21 23 25 25 26 26 26 26 weekly) 5 22 22 23 24 25 25 25 25 6 21 23 25 26 26 26 26 26 Mean 20.3 21.3 22.8 23.7 24.3 24.2 24.5 24.3 SEM 0.6 0.7 0.8 0.8 0.8 0.9 0.7 0.8 10 OBI-888 3 mg/ 1 20 20 22 22 23 23 24 24 kg × 6 2 22 23 23 24 25 24 25 25 IV 3 22 23 24 25 25 25 26 26 (Once 4 22 23 24 26 26 26 26 26 weekly) 5 21 22 23 24 25 24 25 24 6 22 23 25 27 27 27 27 27 Mean 21.5 22.3 23.5 24.7 25.2 24.8 25.5 25.3 SEM 0.3 0.5 0.4 0.7 0.5 0.6 0.4 0.5 P < 0.05 11 MMAE 0.057 mg/ 1 23 24 26 26 27 26 27 27 kg × 6 2 22 22 23 24 25 24 25 25 IV 3 22 22 24 25 25 25 24 24 (Once 4 24 24 25 25 26 25 26 26 weekly) 5 21 22 23 24 25 25 25 24 6 23 24 24 25 25 25 26 25 Mean 22.5 23.0 24.2 24.8 25.5 25.0 25.5 25.2 SEM 0.4 0.4 0.5 0.3 0.3 0.3 0.4 0.5 P < 0.05

TABLE 5-2 Body weight, Xenograft, Breast, MCF-7 in Nude Mice (Day 29-Day 49) Dose (mg/kg) Body Weight (g) Gr. Treatment (Route) No. Day29 Day33 Day36 Day40 Day43 Day46 Day49  1 Vehicle 10 mL/ 1 26 26 26 26 26 26 26 (25 mM Sodium kg × 6 2 28 28 28 28 28 28 29 Citrate, IV 3 27 28 28 28 27 27 27 pH 6.5 + (Once 4 26 26 27 26 26 27 27 100 mM weekly) 5 28 28 28 29 28 28 28 NaCl) 6 24 23 23 23 22 23 24 Mean 26.5 26.5 26.7 26.7 26 26.5 26.8 SEM 0.6 0.8 0.8 0.9 1 0.8 0.7  2 Vehicle 10 mL/ 1 25 25 25 25 26 26 26 (25 mM Sodium kg × 2 2 25 23 23 24 24 23 23 Citrate, IV 3 23 22 24 23 23 24 23 pH 6.5 + (Once 4 28 28 28 28 27 27 28 100 mM weekly) 5 28 29 29 29 29 28 29 NaCl) 6 27 27 28 28 27 28 29 Mean 26.0 25.7 26.2 26.2 26 26.0 26.3 SEM 0.8 1.1 1.0 1.0 1 0.9 1.1 P < 0.05  3 ADC (OBI-999) 10 mg/ 1 26 26 27 27 27 26 28 kg × 2 2 24 24 24 25 24 24 24 IV 3 27 28 28 27 28 27 28 (Once 4 22 22 23 23 23 23 23 weekly) 5 24 24 24 24 25 25 25 6 25 25 25 25 25 25 25 Mean 24.7 24.8 25.2 25.2 25 25.0 25.5 SEM 0.7 0.8 0.8 0.7 1 0.6 0.8 P < 0.05  4 ADC (OBI-999) 0.3 mg/ 1 24 24 25 25 25 25 25 kg × 6 2 26 27 28 27 27 27 28 IV 3 25 25 25 25 25 25 25 (Once 4 25 25 26 26 26 26 27 weekly) 5 25 24 25 25 25 25 25 6 26 26 27 26 26 26 27 Mean 25.2 25.2 26.0 25.7 26 25.7 26.2 SEM 0.3 0.5 0.5 0.3 0 0.3 0.5 P < 0.05  5 ADC (OBI-999) 1 mg/ 1 24 24 24 24 24 24 24 kg × 6 2 26 26 26 26 26 26 27 IV 3 24 25 25 25 25 25 25 (Once 4 27 27 27 27 27 28 28 weekly) 5 26 25 26 26 25 25 26 6 26 26 27 26 25 26 26 Mean 25.5 25.5 25.8 25.7 25.3 25.7 26.0 SEM 0.5 0.4 0.5 0.4 0.4 0.6 0.6  6 ADC (OBI-999) 3 mg/ 1 27 26 27 26 27 27 28 kg × 6 2 24 24 25 24 24 24 24 IV 3 24 24 25 24 24 24 25 (Once 4 26 24 24 25 24 25 27 weekly) 5 23 23 24 24 24 24 24 6 24 24 25 25 25 24 24 Mean 24.7 24.2 25.0 24.7 24.7 24.7 25.3 SEM 0.6 0.4 0.4 0.3 0.5 0.5 0.7 P < 0.05  7 OBI-888 10 mg/ 1 24 25 25 24 24 24 25 kg × 2 2 28 28 28 27 28 28 28 IV 3 24 24 24 24 25 24 25 (Once 4 26 26 26 22 21 22 24 weekly) 5 25 25 26 26 26 26 28 6 25 25 25 25 23 25 24 Mean 25.3 25.5 25.7 24.7 24.5 24.8 25.7 SEM 0.6 0.6 0.6 0.7 1.0 0.8 0.8 P < 0.05  8 OBI-888 0.3 mg/ 1 25 25 25 25 25 25 26 kg × 6 2 23 22 23 22 23 23 24 IV 3 24 25 24 25 24 24 26 (Once 4 24 23 24 24 24 24 24 weekly) 5 24 24 24 24 24 24 24 6 24 24 25 24 25 24 25 Mean 24.0 23.8 24.2 24.0 24.2 24.0 24.8 SEM 0.3 0.5 0.3 0.4 0.3 0.3 0.4 P < 0.05  9 OBI-888 1 mg/ 1 25 25 23 20 24 25 26 kg × 6 2 24 23 24 23 23 23 25 IV 3 22 22 22 23 23 22 22 (Once 4 26 25 26 26 26 26 27 weekly) 5 25 24 25 25 25 25 26 6 27 26 27 27 27 28 27 Mean 24.8 24.2 24.5 24.0 24.7 24.8 25.5 SEM 0.7 0.6 0.8 1.0 0.7 0.9 0.8 10 OBI-888 3 mg/ 1 25 24 25 25 25 24 26 kg × 6 2 26 26 27 26 26 27 28 IV 3 25 25 26 26 26 26 27 (Once 4 27 26 27 26 24 24 24 weekly) 5 25 26 26 26 26 26 27 6 27 26 26 25 25 25 27 Mean 25.8 25.5 26.2 25.7 25.3 25.3 26.5 SEM 0.4 0.3 0.3 0.2 0.3 0.5 0.6 P < 0.05 11 MMAE 0.057 mg/ 1 27 27 27 26 26 26 28 kg × 6 2 25 24 25 25 25 25 26 IV 3 25 24 24 24 22 21 21 (Once 4 26 26 27 28 27 27 25 weekly) 5 24 25 25 26 25 26 26 6 25 26 26 26 26 26 27 Mean 25.3 25.3 25.7 25.8 25.2 25.2 25.5 SEM 0.4 0.5 0.5 0.5 0.7 0.9 1.0 P < 0.05

TABLE 5-3 Body weight, Xenograft, Breast, MCF-7 in Nude Mice (Day 53-Day 77) Dose (mg/kg) Body Weight (g) Gr. Treatment (Route) No. Day53 Day56 Day60 Day63 Day67 Day70 Day74 Day77  1 Vehicle 10 mL/ 1 27 26 27 27 27 27 27 27 (25 mM Sodium kg × 6 2 29 28 28 27 28 28 27 28 Citrate, IV 3 28 27 28 28 27 26 26 26 pH 6.5 + (Once 4 27 27 27 27 28 28 28 28 100 mM weekly) 5 27 28 29 29 28 28 28 27 NaCl) 6 25 26 27 26 26 26 26 25 Mean 27.2 27.0 27.7 27.3 27.3 27.2 27.0 26.8 SEM 0.5 0.4 0.3 0.4 0.3 0.4 0.4 0.5  2 Vehicle 10 mL/ 1 26 26 27 26 27 27 27 27 (25 mM Sodium kg × 2 2 23 21 21 21 21 21 22 22 Citrate, IV 3 24 24 25 25 25 25 25 25 pH 6.5 + (Once 4 28 28 29 28 28 29 29 29 100 mM weekly) 5 28 28 28 28 29 29 29 29 NaCl) 6 28 27 26 26 26 26 27 27 Mean 26.2 25.7 26.0 25.7 26.0 26.2 26.5 26.5 SEM 0.9 1.1 1.2 1.1 1.2 1.2 1.1 1.1 P < 0.05  3 ADC (OBI-999) 10 mg/ 1 28 27 28 27 28 28 27 28 kg × 2 2 24 24 24 24 25 24 24 24 IV 3 28 28 29 28 30 30 29 29 (Once 4 24 23 24 21 23 24 24 25 weekly) 5 25 25 26 24 24 25 26 26 6 25 24 25 26 25 26 23 22 Mean 25.7 25.2 26.0 25.0 25.8 26.2 25.5 25.7 SEM 0.8 0.8 0.9 1.0 1.1 1.0 0.9 1.1 P < 0.05  4 ADC (OBI-999) 0.3 mg/ 1 25 25 26 26 26 26 26 26 kg × 6 2 28 28 28 28 28 28 28 28 IV 3 26 26 26 25 23 24 21 22 (Once 4 28 28 28 27 28 28 28 28 weekly) 5 23 24 26 25 26 27 28 27 6 27 27 29 28 29 27 27 28 Mean 26.2 26.3 27.2 26.5 26.7 26.7 26.3 26.5 SEM 0.8 0.7 0.5 0.6 0.9 0.6 1.1 1.0 P < 0.05  5 ADC (OBI-999) 1 mg/ 1 24 24 24 22 23 23 23 23 kg × 6 2 28 27 29 27 29 29 25 25 IV 3 25 24 25 25 27 26 25 25 (Once 4 28 27 28 28 28 28 29 28 weekly) 5 26 26 27 26 26 27 27 26 6 26 26 27 27 27 27 29 28 Mean 26.2 25.7 26.7 25.8 26.7 26.7 26.3 25.8 SEM 0.7 0.6 0.8 0.9 0.8 0.8 1.0 0.8 P < 0.05  6 ADC (OBI-999) 3 mg/ 1 28 27 28 28 29 29 30 29 kg × 6 2 24 22 22 21 22 21 22 22 IV 3 25 25 26 26 26 26 26 26 (Once 4 26 27 28 28 28 28 28 27 weekly) 5 24 24 25 24 24 25 24 24 6 24 23 24 23 23 23 23 23 Mean 25.2 24.7 25.5 25.0 25.3 25.3 25.5 25.2 SEM 0.7 0.8 1.0 1.2 1.1 1.2 1.3 1.1 P < 0.05  7 OBI-888 10 mg/ 1 25 25 27 26 26 26 26 26 kg × 2 2 28 28 29 29 28 died died died IV 3 24 24 26 21 23 24 24 24 (Once 4 25 27 28 27 27 27 26 25 weekly) 5 27 22 23 27 28 31 35 26 6 23 22 21 24 24 24 22 20 Mean 25.3 24.7 25.7 25.7 26.0 26.4 26.6 24.2 SEM 0.8 1.0 1.3 1.1 0.9 1.3 2.2 1.1 P < 0.05  8 OBI-888 0.3 mg/ 1 26 26 26 26 27 27 28 27 kg × 6 2 24 23 24 25 26 26 27 26 IV 3 26 24 25 25 25 25 26 26 (Once 4 24 24 25 25 25 26 26 25 weekly) 5 24 25 27 25 24 25 25 25 6 26 25 26 26 26 26 26 26 Mean 25.0 24.5 25.5 25.3 25.5 25.8 26.3 25.8 SEM 0.4 0.4 0.4 0.2 0.4 0.3 0.4 0.3 P < 0.05  9 OBI-888 1 mg/ 1 26 26 26 26 25 26 26 24 kg × 6 2 24 25 25 24 26 26 24 25 IV 3 22 27 28 20 21 21 22 21 (Once 4 27 25 26 26 27 27 24 24 weekly) 5 25 26 26 26 26 26 26 26 6 26 19 19 26 26 27 27 27 Mean 25.0 24.7 25.0 24.7 25.2 25.5 24.8 24.5 SEM 0.7 1.2 1.3 1.0 0.9 0.9 0.7 0.8 P < 0.05 10 OBI-888 3 mg/ 1 26 24 26 26 27 27 26 27 kg × 6 2 26 27 27 24 23 21 23 24 IV 3 26 23 23 25 22 25 25 25 (Once 4 24 26 28 23 24 22 23 25 weekly) 5 27 26 27 27 27 27 27 28 6 26 25 27 26 26 27 27 28 Mean 25.8 25.2 26.3 25.2 24.8 24.8 25.2 26.2 SEM 0.4 0.6 0.7 0.6 0.9 1.1 0.7 0.7 P < 0.05 11 MMAE 0.057 mg/ 1 28 26 28 29 27 25 24 23 kg × 6 2 26 26 27 26 26 26 27 27 IV 3 22 23 24 24 25 25 25 24 (Once 4 24 25 26 27 28 28 28 27 weekly) 5 26 24 27 27 27 27 27 28 6 26 27 27 26 28 28 29 29 Mean 25.3 25.2 26.5 26.5 26.8 26.5 26.7 26.3 SEM 0.8 0.6 0.6 0.7 0.5 0.6 0.8 1.0 P < 0.05

FIGS. 4A and 4B show the tumor growth curves in MCF-7 implanted female nude (nu/nu) mice. Intravenous administration of ADC (OBI-999) at 10 mg/kg once weekly for two weeks was associated with significant (T/C value ≤42%) anti-tumor activity from Day 19 to Day 77 compared to the corresponding vehicle control group (FIG. 4A). Furthermore, evidence of a dose-dependent effect was observed in the ADC (OBI-999) treated groups which received once weekly administrations for six weeks. Intravenous administration of ADC (OBI-999) at 0.3 mg/kg once weekly for six weeks was not associated with anti-tumor activity over the course of the study. However, intravenous administration of ADC (OBI-999) at 1 mg/kg and 3 mg/kg once weekly for six weeks was associated with significant (T/C value ≤42%) anti-tumor activity from Day 26 to Day 77 and Day 19 to Day 77, respectively, compared to the corresponding vehicle control group (FIG. 4B).

Intravenous administration of OBI-888 at 10 mg/kg once weekly for two weeks was associated with modest-to-moderate anti-tumor activity both during and for a short time after the dosing phase of the study compared to the corresponding vehicle control group (FIG. 4A). Furthermore, evidence of a dose-dependent effect was observed in the OBI-888 treated groups which received once weekly administrations for six weeks. Intravenous administration of OBI-888 at 0.3 mg/kg once weekly for six weeks was associated with modest anti-tumor activity over the course of the study. Intravenous administration of OBI-888 at 1 mg/kg once weekly for six weeks was associated with moderate anti-tumor activity over the course of the study. Intravenous administration of OBI-888 at 3 mg/kg once weekly for six weeks reached significant (T/C value ≤42%) anti-tumor activity on Day 67 and Day 70, although anti-tumor activity remained close to significant (T/C value ≤42%) as early as Day 26 compared to the corresponding vehicle control group (FIG. 4B).

Intravenous administration of MMAE at 0.057 mg/kg once weekly for six weeks was associated with modest-to-moderate anti-tumor activity both during and for a short time after the dosing phase of the study compared to the corresponding vehicle control group (FIG. 4B).

FIGS. 5A and 5B show the body weight changes in MCF-7 implanted female nude (nu/nu) mice. All test substances at all dose levels were well-tolerated in animals, and were not associated with significant loss in body weight over the course of the study. No overt toxicities were observed during the study period. It also proved the safety of ADC (OBI-999), OBI-888 and MMAE compared to the corresponding vehicle control group.

Example 4: Measurement of the Anti-Tumor Activity of the Exemplary Antibody in Nude Mice (Gastric Cancer)

In a xenograft tumor model of human gastric carcinoma, viable NCI-N87 (ATCC CRL-5822) cells were subcutaneously (SC) implanted (2.5×10⁶ cells/mL with matrigel (1:1) at 0.2 mL/mouse) into the right flank of female nu/nu mice. Tumor implanted mice were divided into seven treatment groups, each group containing eight animals, and one group containing five animals, and dose administrations were initiated one day after cell implantation (denoted as Day 1).

4.1 Test Substances and Dosing Pattern

Test substances ADC (OBI-999), OBI-888, and corresponding vehicle were formulated by diluting stock with a 25 mM sodium citrate, 100 mM NaCl buffer (pH 6.5) and administered intravenously (IV) once weekly for four weeks. Standard agent, MMAE antibody at 0.191 mg/kg, and corresponding vehicle (PBS pH 7.4) were administered intraperitoneally (IP) once weekly for four weeks. One treatment group received combination therapy of test substance, OBI-888 at 10 mg/kg, with MMAE at 0.191 mg/kg.

TABLE 6 Study Design for Anti-Tumor Activity of the exemplary antibody in Nude Mice (Gastric cancer) Mice^(c, d) Dosage (nu/nu) Group Test Compound Route mL/kg mg/kg (female) 1 Vehicle^(a) + IP + IV 10 N/A 8 Vehicle^(b) 2 ADC (OBI-999)^(b) IV 10 1 8 3 ADC (OBI-999)^(b) IV 10 3 8 4 ADC (OBI-999)^(b) IV 10 10 8 5 OBI-888^(b) IV 10 10 8 6 Anti-CD30 ADC^(b) IV 10 3 5 (OBI-910) 7 MMAE^(a) + IP + IV 10 0.191 + 10 8 OBI-888^(b) 8 MMAE^(a) IP 10 0.191 8 ^(a)PBS, pH 7.4 (high concentration of MMAE will be stored in 100% DMSO and then is diluted with PBS, pH 7.4) ^(b)25 mM Sodium Citrate + 100 mM NaCl, pH 6.5 ^(c)Vehicle and test substances are administered once weekly for four weeks starting one day after tumor cell implantation (denoted as Day 1). ^(d)NCI-N87 at 2.5 × 10⁶ cells/mouse with matrigel (1:1) in 200 uL are injected subcutaneously into right flank of female nu/nu mice. Tumor size/body weight monitoring: twice weekly till Day 70 or the study is terminated when mean tumor volume in the vehicle control group reaches 2000 mm³. Pictures are required to be taken at sacrifice.

4.2 Cell Line

Viable human gastric carcinoma NCI-N87 (ATCC CRL-5822) cell line was purchased and cultured in Eurofins Panlabs Taiwan, Ltd. The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37° C. in 5% CO₂ incubator and implanted subcutaneously in the right flank of each mouse. 4.3 Animals

Female nude (nu/nu) mice aged 5-6 weeks obtained from BioLasco Taiwan (under Charles River Laboratories Licensee) were used. The animals were housed in individually ventilated cages (IVC, 36 Mini Isolator system). The allocation for 3 animals was 27×20×14 in cm. All animals were maintained in a hygienic environment under controlled temperature (20-24° C.) and humidity (30%-70%) with 12-hour light/dark cycle. Free access to standard lab diet [MFG (Oriental Yeast Co., Ltd., Japan)] and autoclaved tap water in bottles were granted. All aspects of this work including housing, experimentation, and animal disposal were performed in general accordance with the “Guide for the Care and Use of Laboratory Animals: Eighth Edition” (National Academies Press, Washington, D.C., 2011) in our AAALAC-accredited laboratory animal facility. In addition, the animal care and use protocol was reviewed and approved by the IACUC at Eurofins Panlabs Taiwan, Ltd.

4.4 Chemicals

0.9% NaCl (Sin-Tong, Taiwan), Fetal bovine serum (HyClone, USA), Matrigel (BD, USA) and RPMI-1640 (HyClone, USA).

4.5 Equipment

Animal cage (Tecniplast, Italy), Beaker 1000 mL (Kimax, USA), Calipers (Mitutoyo, Japan), Class II biological safety cabinet (NuAire, USA), Individually ventilated cages (IVC, 36 Mini Isolator system) (Tecniplast, Italy), Mouse scale #Z-40 (Taconic, USA), Stainless forceps (Klappenecker, Germany) and Vertical laminar flow (Tsao-Hsin, Taiwan). 4.6 Methods

The tumor volumes, body weights, mortality, and signs of overt toxicity were monitored and recorded twice weekly for 100 days. Tumor growth inhibition was calculated as T/C (treatment/control)×100%. A T/C value ≤42% compared to that of the vehicle control group was considered significant anti-tumor activity. Two-way ANOVA followed by Bonferroni test was used to ascertain the statistically significant significance of groups compared to respective vehicle control (*p<0.05).

4.7 Results

TABLE 7-1 Tumor volume, Xenograft, Gastric, NCI-N87 in Female nu/nu Mice (Day 1-Day 25) Dose (mg/kg) Tumor Volume (mm³) Gr. Treatment (Route) No. Day1 Day4 Day8 Day11 Day15 Day18 Day22 Day25 1 Vehicle 10 mL/ 1  93 100 137 258 372 453 635 613 (PBS, pH 7.4) kg × 4 2 118 117 131 166 175 216 219 225 + (Once 3 108 141 177 333 392 432 600 704 Vehicle weekly) 4  99 123 146 332 332 375 442 498 (25 mM IP 5 103 157 162 289 292 335 455 493 Sodium Citrate, + 6  96 124 146 303 325 514 560 664 + IV 7  86 106 144 268 271 321 329 489 100 mM 8  98 123 133 296 344 406 510 510 NaCl, Mean 100 124 147 281 313 382 469 525 pH 6.5) SEM  3  6  5  19  24  32  50  52 2 ADC (OBI-999) 1 mg/ 1  85  98 110 152  92 104 108 115 kg × 4 2  88 112  99 131 117 141 139 159 IV 3  93 113  97 144 129 143 169 208 (Once 4  94 119  88 176 119 103  77 121 weekly) 5 103 117 103 104 113 113  85  80 6  88  97  83 144 131 131 139 145 7 103 104  96 135 121 131 143 150 8 101 123  94  97  88  78  91 133 Mean  94 110  96 135 114  118*  119*  139* SEM  3  3  3  9  6  8  12  13 % T/C —  89  65  48  36#  31#  25#  26# % TGI —  11  35  52  64  69  75  74 3 ADC (OBI-999) 3 mg/ 1  80 102 121  91  60  60  68  73 kg × 4 2  96 131  89  91  79  79  79  65 IV 3  97 125  89  96  99  79  69  66 (Once 4  97  93  71  93  94  86  77  76 weekly) 5  90 131  80  89  84  77  53  57 6 127 160  77  81  91  70  43  68 7  94 127 101  87 108  85  79  77 8  77  88  60  72  93  99  69  70 Mean  95 120  86  88  89  79*  67*  69* SEM  5  8  7  3  5  4  5  2 % T/C —  97  59  31#  28#  21#  14#  13# % TGI —  3  41  69  72  79  86  87 4 ADC (OBI-999) 10 mg/ 1  93  89  82  66  61  86  54  68 kg × 4 2 110 115  97  85  72  71  51  73 IV 3  88 125  85  86  93  51  41  58 (Once 4  94 104 101  86  93  89  73  85 weekly) 5  96  86  73  81  74  40  41  69 6  87 127  96 104 101  86  53  57 7  82 108 110  82  86  73  70  70 8  96 115  88  85  77  68  66  62 Mean  93 109  92  84  82  71*  56*  68* SEM  3  5  4  4  5  6  4  3 % T/C —  88  63  30#  26#  19#  12#  13# % TGI —  12  37  70  74  81  88  87 5 OBI-888 10 mg/ 1  94 106 117 179 214 248 356 358 kg × 4 2 101 133 157 272 318 321 409 394 IV 3  94 104 114 199 238 295 307 396 (Once 4  78 135 125 150 281 426 455 460 weekly) 5 123 150 144 236 252 458 522 551 6  91 111 115 195 256 279 401 401 7  94 111 106 211 233 348 359 432 8  86 113  89 144 216 288 385 467 Mean  95 120 121 198 251 333 399 432 SEM  5  6  8  15  12  26  23  21 % T/C —  97  82  70  80  87  85  82 % TGI —  3  18  30  20  13  15  18 6 Anti-CD30 ADC 3 mg/ 1 108 110 112 101  91  91  97 104 (OBI-910) kg × 4 2  97  81  94 121 117 121 129 130 IV 3  78  94 111 125 106 110 129 166 (Once 4 117  89  94 108 127 128 155 133 weekly) 5 111 121 111 127 129 146 172 174 SEM 102  99 104 116 114  119*  136*  141* Mean  7  7  4  5  7  9  13  13 % T/C —  80  71  41#  36#  31#  29#  27# % TGI —  20  29  59  67  69  71  73 7 MMAE 0.191 mg/ 1  99 104 146 164 214 214 222 243 + kg × 4 2 111 121 112 146 161 211 243 269 OBI-888 IP 3 121  88 103 103 159 145 119 130 (Once 4 112  99 119 119 163 233 320 415 weekly) 5  83 125 112 146 164 186 237 236 + 6  87  74 died died died died died died 10 mg/ 7  81  99 111 132 181 214 239 269 kg × 4 8  78 104 108 113 192 179 181 217 IV Mean  97 102 116 132 176 197 223  254* (Once SEM  6  6  5  8  8  11  23  32 weekly) % T/C —  82  79  47  56  52  48  48 % TGI —  18  21  53  44  48  52  52 8 MMAE 0.191 mg/ 1  61 106 117 162 153 152 144 146 kg × 4 2  89 102 142 158 189 213 201 216 IP 3  83 115 127 137 178 234 246 259 (Once 4  88 115 115 169 231 255 303 356 weekly) 5 125 115 110 174 175 231 252 315 6  88 died died died died died died died 7 110 104 125 187 208 228 322 353 8 121 109 119 166 189 211 296 296 Mean  96 109 122 165 189 218 252 277 SEM  8  2  4  6  9  12  24  29 % T/C —  88  83  59  60  57  54  53 % TGI —  12  17  41  40  43  46  47

TABLE 7-2 Tumor volume, Xenograft, Gastric, NCI-N87 in Female nu/nu Mice (Day 29-Day 53) Dose (mg/kg) Tumor Volume (mm³) Gr. Treatment (Route) No. Day29 Day32 Day36 Day39 Day43 Day46 Day50 Day53 1 Vehicle 10 mL/kg × 4 1  645  706  853  926 1062 1069 1116 1127 (PBS, pH 7.4) (Once 2  299  299  416  432  451  455  484  513 + weekly) 3  779 1079 1355 1479 1592 1862 2039 2546 Vehicle IP 4  623  628  719  756  792  792  798  811 (25 mM + 5  702  864  895 1201 1309 1553 1800 2004 Sodium Citrate, IV 6  862  956 1034 1135 1236 1420 1849 2009 + 7  489  489  503  508  564  630  653  719 100 mM NaCl, 8  665  707  746  828  863  900  968 1036 pH 6.5) Mean  633  716  815  908  984 1085 1213 1346 SEM  62  89  105  125  137  172  212  262 2 ADC (OBI-999) 1 mg/ 1  113  73  72  72  64  58  53  51 kg × 4 2  164  192  228  234  258  299  324  346 IV 3  222  240  243  252  275  310  345  345 (Once 4  121  121  121  125  125  113 died died weekly) 5  94  110  112  97  96  96  104  108 6  145  148  152  168  183  202  208  225 7  152  176  184  199  216  218  248  271 8  133  133  137  148  152  187  208  231 Mean  143*  149*  156*  162*  171*  185*  213*  225* SEM  14  19  21  23  27  33  40  42 % T/C   23#   21#   19#   18#   17#   17#   18#   17# % TGI  77  79  81  82  83  83  82  83 3 ADC (OBI-999) 3 mg/ 1  56  72  72  66  64  63  60  58 kg × 4 2  68  68  73  76  72  64  61  59 IV 3  59  40  41  43  38  34  33  32 (Once 4  54  48  44  36  38  38  38  38 weekly) 5  88  32   0   0   0   0   0   0 6  64  64  62  56  55  54  52  50 7  104  89  85  82  76  72  69  36 8  70  69  66  65  62  60  60  57 Mean   70*   60*   55*   53*   51*   48*   47*   42* SEM   6   7   9   9   9   8   8   8 % T/C   11#    8#    7#    6#    5#    4#    4#    3# % TGI  89  92  93  94  95  96  96  97 4 ADC (OBI-999) 10 mg/ 1  66  61  51  51  50  49  48  47 kg × 4 2  56  57  64  61  59  59  58  58 IV 3  44  47  46  40  38   0   0   0 (Once 4  77  69  65  65  64  64  61  61 weekly) 5  52  59  56  54  52  51  49  46 6  70  59  53  53  53  52  50  49 7  67  68  68  62  60  60  60  57 8  66  77  66  61  60  57  55  54 Mean   62*   62*   59*   56*   55*   49*   48*   47* SEM   4   3   3   3   3   7   7   7 % T/C   10#   9#   7#   6#   6#   5#   4#   3# % TGI  90  91  93  94  94  95  96  97 5 OBI-888 10 mg/ 1  418  583  605  612  698  801  819  926 kg × 4 2  590  689  694  694  773  845 1016 1074 IV 3  460  466  508  588  668  770  828 1030 (Once 4  714  830  859 1040 1103 1359 1614 1885 weekly) 5  739  744  835  886  968 1230 1238 1342 6  565  565  652  723  840  979 1012 1074 7  530  728  780  780  900 1057 1072 1258 8  533  652  719  722  869  958 1065 1065 Mean  569  657  707  756  852 1000 1083 1207 SEM  40  41  42  52  51  74  90  107 % T/C  90  92  87  83  87  92  89  90 % TGI  10   8  13  17  13   8  11  10 6 Anti-CD30 ADC 3 mg/ 1  181  187  192  179  171  164  148  141 (OBI-910) kg × 4 2  208  231  189  191  210  256  292  320 IV 3  225  243  243  246  252  296  327  355 (Once 4  197  207  217  217  217  259  262  262 weekly) 5  282  272  377  381  411  546  546  579 Mean  219*  228*  244*  243*  252*  304*  315*  331* SEM  17  15  35  36  42  64  65  72 % T/C   35#   32#   30#   27#   26#   28#   26#   25# % TGI  65  68  70  73  74  72  74  75 7 MMAE 0.191 mg/ 1  293  292  356  394  407  509  562  578 + kg × 4 2  286  272  279  293  352  356  385  407 OBI-888 IP 3  143  189  199  159  156  164  166  192 (Once 4  465  465  469  484  484  515  538  614 weekly) 5  283  325  387  405  417  458  476  521 + 6 died died died died died died died died 10 mg/ 7  325  405  515  514  540  617  688  819 kg × 4 8  314  293  289  292  295  360  372  432 IV Mean  301*  320*  356*  363*  379*  426*  455*  509* (Once SEM  36  34  42  47  48  56  63  74 weekly) % T/C  48  45  44   40#   39#   39#   38#   38# % TGI  52  55  56  60  61  61  62  62 8 MMAE 0.191 mg/ 1  162  160  94  94  97  91  89  88 kg × 4 2  277  318  345  385  414  606  623  682 IP 3  399  397  390  407  429  535  569  590 (Once 4  406  385  389  442  489  495  550  581 weekly) 5  439  446  506  530  581  719  766  816 6 died died died died died died died died 7  525  584  658  671  780  878  936 1094 8  387  432  459  487  549  561  590  620 Mean  371*  389*  406*  431*  477*  555*  589*  639* SEM  44  49  65  67  78  92  98  114 % T/C  59  54  50  47  48  51  49  47 % TGI  41  46  50  53  52  49  51  53

TABLE 7-3 Tumor volume, Xenograft, Gastric, NCI-N87 in Female nu/nu Mice (Day 57-Day 85) Dose/Route Tumor Volume (mm³) Gr. Treatment (mg/kg) No. Day57 Day60 Day 64 Day67 Day70 Day74 Day78 Day81 Day85 1 Vehicle 10 mL/ 1 NA NA NA NA NA NA NA NA NA (PBS, pH 7.4) kg × 4 2 NA NA NA NA NA NA NA NA NA + (Once 3 NA NA NA NA NA NA NA NA NA Vehicle weekly) 4 NA NA NA NA NA NA NA NA NA (25 mM Sodium Citrate, IP + IV 5 NA NA NA NA NA NA NA NA NA + 6 NA NA NA NA NA NA NA NA NA 100 mM 7 NA NA NA NA NA NA NA NA NA NaCl, pH 6.5) 8 NA NA NA NA NA NA NA NA NA Mean — — — — — — — — — SEM — — — — — — — — — 2 ADC (OBI-999) 1 mg/ 1 48 48 47 44 36 35 35 37 37 kg × 4 2 386 417 426 471 496 519 528 553 567 IV 3 373 424 456 536 556 578 630 690 760 (Once 4 died died died died died died died died died weekly) 5 104 104 101 101 94 91 97 97 94 6 231 206 223 229 254 277 292 298 306 7 328 396 455 521 544 593 658 684 778 8 251 309 347 489 529 570 680 717 833 Mean 246 272 294 342 358 380 417 439 482 SEM 49 58 65 80 85 92 103 110 127 % T/C — — — — — — — — — 3 ADC (OBI-999) 3 mg/ 1 55 55 53 53 54 55 57 60 62 kg × 4 2 56 56 56 59 61 64 65 68 70 IV 3 30 29 29 27 27 26 25 25 24 (Once 4 39 40 41 41 45 51 54 59 64 weekly) 5 0 0 0 0 0 0 0 0 0 6 50 51 53 55 58 60 63 63 64 7 died died died died died died died died died 8 55 55 57 60 60 57 55 53 52 Mean 41 41 41 42 44 45 46 47 48 SEM 8 8 8 8 9 9 9 9 10 % T/C — — — — — — — — — 4 ADC (OBI-999) 10 mg/ 1 46 46 49 51 54 54 52 52 52 kg × 4 2 55 58 55 55 53 51 48 48 51 IV 3 0 0 0 0 0 0 0 0 0 (Once 4 61 61 64 65 65 65 62 60 57 weekly) 5 45 45 45 44 42 40 38 38 36 6 46 45 45 45 44 44 42 40 40 7 57 57 60 62 62 57 55 53 40 8 54 53 51 51 48 46 45 45 45 Mean 46 46 46 47 46 45 43 42 40 SEM 7 7 7 7 7 7 7 7 6 % T/C — — — — — — — — — 5 OBI-888 10 mg/ 1 1005 1145 1152 1220 1281 NA NA NA NA kg × 4 2 1135 1369 1406 1458 1458 NA NA NA NA IV 3 1048 1090 1146 1331 1371 NA NA NA NA (Once 4 2137 2313 2334 2669 2692 NA NA NA NA weekly) 5 1429 1475 1483 1491 1491 NA NA NA NA 6 1324 1371 1433 1571 1694 NA NA NA NA 7 1302 1378 1468 1617 1628 NA NA NA NA 8 1310 1371 1415 1553 1580 NA NA NA NA Mean 1336 1439 1480 1614 1649 — — — — SEM 126 133 131 158 156 — — — — % T/C — — — — — — — — — 6 Anti-CD30 ADC 3 mg/ 1 135 133 127 123 119 117 115 113 113 (OBI-910) kg × 4 2 360 437 467 610 631 733 862 905 999 IV 3 427 453 503 634 634 706 854 928 1006 (Once 4 269 352 368 415 411 485 515 539 559 weekly) 5 584 605 611 633 645 689 689 729 749 Mean 355 396 415 483 488 546 607 643 685 SEM 75 77 82 99 102 116 138 150 166 % T/C — — — — — — — — — 7 MMAE 0.191 mg/ 1 640 701 721 814 841 930 969 1065 1175 + kg × 4 2 461 490 510 540 551 584 623 623 623 OBI-888 IP 3 199 206 200 228 234 240 248 255 276 (Once 4 663 663 677 723 728 743 757 767 772 weekly) 5 567 681 708 796 808 951 958 965 1064 + 6 died died died died died died died died died 10 mg/ 7 845 897 897 965 1044 1051 1111 1171 1171 kg × 4 8 436 409 409 404 420 467 477 477 482 IV Mean 544 578 589 639 661 709 735 760 795 (Once SEM 77 86 88 98 104 111 116 125 134 weekly) % T/C — — — — — — — — — 8 MMAE 0.191 mg/ 1 85 83 82 79 76 74 74 77 77 kg × 4 2 694 745 765 883 943 1012 1042 1064 1097 IP 3 663 783 788 827 870 909 955 961 1033 (Once 4 627 664 702 726 726 856 890 903 933 weekly) 5 870 854 920 1070 1090 1117 1197 1197 1331 6 died died died died died died died died died 7 1141 1234 1272 1300 1358 1422 1431 1517 1558 8 650 676 703 840 859 916 928 935 988 Mean 676 720 747 818 846 901 931 951 1002 SEM 120 129 134 143 150 156 160 166 175 % T/C — — — — — — — — —

TABLE 7-4 Tumor volume, Xenograft, Gastric, NCI-N87 in Female nu/nu Mice (Day 88-Day 100) Dose (mg/kg) Tumor Volume (mm³) Gr. Treatment (Route) No. Day88 Day91 Day95 Day98 Day100 1 Vehicle 10 mL/kg × 4 1 NA NA NA NA NA (PBS, pH 7.4) + (Once weekly) 2 NA NA NA NA NA Vehicle IP + IV 3 NA NA NA NA NA (25 mM 4 NA NA NA NA NA Sodium Citrate, + 5 NA NA NA NA NA 100 mM NaCl, 6 NA NA NA NA NA pH 6.5) 7 NA NA NA NA NA 8 NA NA NA NA NA Mean — — — — — SEM — — — — — 2 ADC (OBI-999) 1 mg/kg × 4 1 36 36 34 34 34 IV 2 588 719 817 832 881 (Once weekly) 3 814 841 868 898 959 4 died died died died died 5 91 91 94 94 96 6 306 298 295 292 289 7 802 817 866 942 996 8 834 924 1163 1284 1338 Mean 496 532 591 625 656 SEM 132 143 167 182 193 % T/C — — — — — 3 ADC (OBI-999) 3 mg/kg × 4 1 65 65 68 70 73 IV 2 70 70 68 65 62 (Once weekly) 3 23 22 21 21 0 4 66 72 76 85 117 5 0 0 0 0 0 6 65 65 65 57 55 7 died died died died died 8 52 52 51 50 49 Mean 49 49 50 50 51 SEM 10 10 11 11 16 % T/C — — — — — 4 ADC (OBI-999) 10 mg/kg × 4 1 52 50 49 48 47 IV 2 53 55 55 53 51 (Once weekly) 3 0 0 0 0 0 4 55 55 55 54 53 5 36 36 36 35 28 6 40 40 37 34 0 7 NA NA NA NA NA 8 43 42 40 37 24 Mean 40 40 39 37 29 SEM 7 7 7 7 9 % T/C — — — — — 5 OBI-888 10 mg/kg × 4 1 NA NA NA NA NA IV 2 NA NA NA NA NA (Once weekly) 3 NA NA NA NA NA 4 NA NA NA NA NA 5 NA NA NA NA NA 6 NA NA NA NA NA 7 NA NA NA NA NA 8 NA NA NA NA NA Mean — — — — — SEM — — — — — % T/C — — — — — 6 Anti-CD30 ADC 3 mg/kg × 4 1 112 110 106 103 99 (OBI-910) IV 2 1038 1183 1347 1408 1455 (Once weekly) 3 1014 1081 1176 1236 1311 4 573 597 657 693 719 5 804 888 895 856 942 Mean 708 772 836 859 905 SEM 171 193 217 228 240 % T/C — — — — — 7 MMAE + 0.191 mg/kg × 4 1 1253 1329 1466 1595 1732 OBI-888 IP 2 628 628 640 646 628 (Once weekly) + 3 290 293 296 296 296 10 mg/kg × 4 4 788 820 815 810 753 IV 5 1087 1122 1226 1284 1301 (Once weekly) 6 died died died died died 7 1171 1208 1217 1225 1242 8 468 454 436 408 386 Mean 812 836 871 895 905 SEM 140 150 167 184 201 % T/C — — — — — 8 MMAE 0.191 mg/kg × 4 1 74 72 68 68 62 IP 2 1104 1122 1192 1215 1254 (Once weekly) 3 1117 1130 1184 1265 1273 4 933 933 947 1061 1076 5 1346 1346 1354 1398 1444 6 died died died died died 7 1626 1636 1646 1656 1590 8 1023 1023 1029 1043 1043 Mean 1032 1037 1060 1101 1106 SEM 182 183 186 189 189 % T/C — — — — —

TABLE 8-1 Body Weight, Xenograft, Gastric, NCI-N87 in Female nu/nu Mice (Day 1-Day 25) Dose (mg/kg) Body Weight (g) Gr. Treatment (Route) No. Day1 Day4 Day8 Day11 Day15 Day18 Day22 Day25 1 Vehicle 10 mL/kg × 4 1 22 24 24 25 24 24 25 25 (PBS, pH 7.4) + (Once weekly) 2 23 24 25 25 24 24 24 25 Vehicle IP + IV 3 21 22 23 24 24 24 24 25 (25 mM 4 22 24 22 22 23 24 24 24 Sodium Citrate, + 5 22 23 24 25 26 26 26 25 100 mM NaCl, 6 23 24 25 26 25 26 25 25 pH 6.5) 7 23 23 24 25 24 25 25 24 8 24 24 25 26 25 25 25 26 Mean 22.5 23.5 24.0 24.8 24.4 24.8 24.8 24.9 SEM 0.3 0.3 0.4 0.5 0.3 0.3 0.3 0.2 2 ADC (OBI-999) 1 mg/kg × 4 1 22 23 23 24 24 24 24 24 IV 2 22 24 25 25 24 24 24 24 (Once weekly) 3 21 22 23 24 23 23 23 23 4 22 23 24 24 23 23 22 24 5 23 23 26 26 27 27 27 27 6 20 22 23 23 24 24 24 24 7 22 22 24 25 24 23 24 24 8 23 23 24 24 24 24 24 24 Mean 21.9 22.8 24.0 24.4 24.1 24.0 24.0 24.3 SEM 0.4 0.3 0.4 0.3 0.4 0.5 0.5 0.4 3 ADC (OBI-999) 3 mg/kg × 4 1 22 23 23 24 23 24 24 24 IV 2 21 22 23 24 25 25 25 26 (Once weekly) 3 23 22 22 23 22 23 24 24 4 23 23 23 22 22 23 24 23 5 23 24 25 24 24 24 24 24 6 22 22 23 24 24 24 24 24 7 21 22 23 24 25 25 25 25 8 22 22 21 21 22 22 23 22 Mean 22.1 22.5 22.9 23.3 23.4 23.8 24.1 24.0 SEM 0.3 0.3 0.4 0.4 0.5 0.4 0.2 0.4 4 ADC (OBI-999) 10 mg/kg × 4 1 22 22 22 22 23 24 23 24 IV 2 21 21 23 23 24 23 24 24 (Once weekly) 3 22 23 23 22 22 23 23 24 4 21 21 22 22 23 23 23 23 5 23 24 24 24 23 23 24 24 6 22 23 24 24 24 24 24 24 7 21 22 22 23 22 23 23 22 8 23 23 25 26 26 25 26 26 Mean 21.9 22.4 23.1 23.3 23.4 23.5 23.8 23.9 SEM 0.3 0.4 0.4 0.5 0.5 0.3 0.4 0.4 5 OBI-888 10 mg/kg × 4 1 21 21 22 23 24 24 23 24 IV 2 22 23 24 24 25 25 25 26 (Once weekly) 3 19 19 19 20 21 22 22 23 4 22 22 22 23 23 23 23 24 5 21 22 23 23 22 22 23 23 6 21 21 22 23 23 23 24 24 7 20 22 22 22 21 21 22 22 8 21 21 22 22 21 21 22 22 Mean 20.9 21.4 22.0 22.5 22.5 22.6 23.0 23.5 SEM 0.4 0.4 0.5 0.4 0.5 0.5 0.4 0.5 6 Anti-CD30 ADC 3 mg/kg × 4 1 21 22 23 23 23 24 24 22 (OBI-910) IV 2 20 21 22 22 22 23 23 22 (Once weekly) 3 21 22 22 23 22 23 24 24 4 22 22 22 23 24 24 25 25 5 22 23 23 23 24 25 25 26 Mean 21.2 22.0 22.4 22.8 23.0 23.8 24.2 23.8 SEM 0.4 0.3 0.2 0.2 0.4 0.4 0.4 0.8 7 MMAE + 0.191 mg/kg × 4 1 20 18 22 21 23 24 24 23 OBI-888 IP 2 21 20 22 22 21 22 23 23 (Once weekly) + 3 21 20 22 22 22 22 22 22 10 mg/kg × 4 4 21 20 22 23 23 22 23 22 IV 5 23 21 24 24 25 24 25 26 (Once weekly) 6 23 21 died died died died died died 7 22 22 24 23 24 23 24 24 8 22 21 23 23 23 24 24 25 Mean 21.6 20.4 22.7 22.6 23.0 23.0 23.6 23.6 SEM 0.4 0.4 0.4 0.4 0.5 0.4 0.4 0.6 8 MMAE 0.191 mg/kg × 4 1 20 18 22 22 24 24 24 24 IP 2 20 19 22 22 23 22 22 22 (Once weekly) 3 22 22 22 23 23 23 24 24 4 24 21 23 25 25 26 25 25 5 22 20 23 24 24 24 25 24 6 21 died died died died died died died 7 24 24 24 23 23 24 25 25 8 22 19 19 19 22 22 24 24 Mean 21.9 20.4 22.1 22.6 23.4 23.6 24.1 24.0 SEM 0.5 0.8 0.6 0.7 0.4 0.5 0.4 0.4

TABLE 8-2 Body Weight, Xenograft, Gastric, NCI-N87 in Female nu/nu Mice (Day 29-Day 53) Dose (mg/kg) Body Weight (g) Gr. Treatment (Route) No. Day29 Day32 Day36 Day39 Day43 Day46 Day50 Day 53 1 Vehicle 10 mL/kg × 4 1 27 26 26 27 26 27 27 29 (PBS, pH 7.4) + (Once weekly) 2 26 26 26 27 26 27 28 29 Vehicle IP + IV 3 26 27 27 27 27 27 28 28 (25 mM 4 25 25 26 26 26 26 27 28 Sodium Citrate, + 5 27 27 28 28 28 29 29 29 100 mM NaCl, 6 25 25 26 26 26 27 25 27 pH 6.5) 7 25 26 26 27 26 26 26 27 8 27 27 27 27 27 27 27 28 Mean 26.0 26.1 26.5 26.9 26.5 27.0 27.1 28.1 SEM 0.3 0.3 0.3 0.2 0.3 0.3 0.4 0.3 2 ADC (OBI-999) 1 mg/kg × 4 1 24 24 26 25 25 25 25 25 IV 2 26 26 26 27 26 27 26 27 (Once weekly) 3 24 24 25 26 26 26 26 26 4 23 24 24 23 20 19 died died 5 28 28 28 29 29 30 29 29 6 25 25 26 27 27 28 27 28 7 25 25 26 26 25 26 26 28 8 26 25 26 27 26 27 27 28 Mean 25.1 25.1 25.9 26.3 25.5 26.0 26.6 27.3 SEM 0.5 0.5 0.4 0.6 0.9 1.1 0.5 0.5 3 ADC (OBI-999) 3 mg/kg × 4 1 25 25 26 26 26 27 27 28 IV 2 27 27 29 29 25 24 26 27 (Once weekly) 3 24 24 25 26 26 27 26 27 4 24 24 24 25 25 25 25 26 5 26 25 26 27 27 27 27 27 6 25 25 26 26 26 26 26 27 7 26 26 25 24 23 21 18 17 8 23 23 24 25 23 24 24 25 Mean 25.0 24.9 25.6 26.0 25.1 25.1 24.9 25.5 SEM 0.5 0.4 0.6 0.5 0.5 0.7 1.0 1.3 4 ADC (OBI-999) 10 mg/kg × 4 1 24 25 26 26 26 26 27 28 IV 2 25 25 25 25 26 25 26 26 (Once weekly) 3 24 25 25 26 26 26 26 26 4 24 25 25 26 25 26 26 26 5 25 24 25 26 26 26 26 26 6 26 26 27 28 27 27 27 27 7 23 24 25 25 24 26 25 25 8 28 28 27 28 27 28 28 29 Mean 24.9 25.3 25.6 26.3 25.9 26.3 26.4 26.6 SEM 0.5 0.5 0.3 0.4 0.4 0.3 0.3 0.5 5 OBI-888 10 mg/kg × 4 1 24 25 25 26 26 27 26 27 IV 2 27 25 27 28 28 29 29 30 (Once weekly) 3 23 23 24 26 25 26 25 27 4 25 25 26 27 27 27 27 28 5 24 24 24 24 24 25 25 26 6 25 25 26 26 27 26 26 27 7 23 23 23 24 24 25 25 26 8 22 23 24 24 25 25 25 26 Mean 24.1 24.1 24.9 25.6 25.8 26.3 26.0 27.1 SEM 0.5 0.4 0.5 0.5 0.5 0.5 0.5 0.5 6 Anti-CD30 ADC 3 mg/kg × 4 1 23 24 25 27 26 26 25 26 (OBI-910) IV 2 22 23 24 25 25 26 26 27 (Once weekly) 3 25 25 25 26 26 27 27 28 4 26 26 26 26 26 27 27 28 5 26 26 27 28 27 27 26 25 Mean 24.4 24.8 25.4 26.4 26.0 26.6 26.2 26.8 SEM 0.8 0.6 0.5 0.5 0.3 0.2 0.4 0.6 7 MMAE + 0.191 mg/kg × 4 1 24 24 24 26 26 26 26 27 OBI-888 IP 2 23 23 24 24 24 24 24 25 (Once weekly) + 3 23 23 23 24 22 23 23 25 10 mg/kg × 4 4 24 24 25 25 24 25 24 25 IV 5 28 27 27 29 28 29 29 30 (Once weekly) 6 died died died died died died died died 7 25 25 26 26 25 26 25 26 8 25 24 25 26 25 26 26 27 Mean 24.6 24.3 24.9 25.7 24.9 25.6 25.3 26.4 SEM 0.6 0.5 0.5 0.6 0.7 0.7 0.7 0.7 8 MMAE 0.191 mg/kg × 4 1 25 25 26 26 26 26 26 27 IP 2 24 23 24 24 24 25 24 24 (Once weekly) 3 25 24 25 26 26 27 26 27 4 26 27 25 26 26 27 26 27 5 26 26 26 26 26 27 27 27 6 died died died died died died died died 7 25 26 25 26 26 26 26 26 8 26 25 25 26 26 26 27 28 Mean 25.3 25.1 25.1 25.7 25.7 26.3 26.0 26.6 SEM 0.3 0.5 0.3 0.3 0.3 0.3 0.4 0.5

TABLE 8-3 Body Weight, Xenograft, Gastric, NCI-N87 in Female nu/nu Mice (Day 57-Day 85) Dose (mg/kg) Body Weight (g) Gr. Treatment (Route) No. Day57 Day60 Day64 Day67 Day70 Day74 Day78 Day81 Day85 1 Vehicle 10 mL/kg 4 1 NA NA NA NA NA NA NA NA NA (PBS, pH 7.4) + (Once weekly) 2 NA NA NA NA NA NA NA NA NA Vehicle IP + IV 3 NA NA NA NA NA NA NA NA NA (25 mM 4 NA NA NA NA NA NA NA NA NA Sodium Citrate, + 5 NA NA NA NA NA NA NA NA NA 100 mM NaCl, 6 NA NA NA NA NA NA NA NA NA pH 6.5) 7 NA NA NA NA NA NA NA NA NA 8 NA NA NA NA NA NA NA NA NA Mean — — — — — — — — — SEM — — — — — — — — — 2 ADC (OBI-999) 1 mg/kg × 4 1 25 25 26 26 26 26 26 26 27 IV 2 27 28 28 28 28 28 28 28 29 (Once weekly) 3 27 27 27 27 27 28 28 29 30 4 died died died died died died died died died 5 29 28 28 30 28 29 28 28 29 6 28 28 28 28 28 30 29 29 30 7 27 26 25 26 25 27 29 28 27 8 28 28 28 29 28 28 29 29 30 Mean 27.3 27.1 27.1 27.7 27.1 28.0 28.1 28.1 28.9 SEM 0.5 0.5 0.5 0.6 0.5 0.5 0.4 0.4 0.5 3 ADC (OBI-999) 3 mg/kg × 4 1 27 28 27 27 28 28 28 28 28 IV 2 27 28 29 29 29 29 30 31 31 (Once weekly) 3 26 27 27 27 27 28 27 27 27 4 26 26 26 26 26 26 27 26 27 5 27 27 27 27 27 27 27 27 28 6 26 27 26 26 26 27 27 27 28 7 died died died died died died died died died 8 25 26 25 26 25 26 26 26 26 Mean 26.3 27.0 26.7 26.9 26.9 27.3 27.4 27.4 27.9 SEM 0.3 0.3 0.5 0.4 0.5 0.4 0.5 0.6 0.6 4 ADC (OBI-999) 10 mg/kg × 4 1 27 28 28 29 28 29 29 28 29 IV 2 27 27 28 28 28 28 27 28 26 (Once weekly) 3 26 28 27 27 28 26 26 25 26 4 27 25 26 28 27 27 28 27 28 5 27 27 26 27 27 27 26 26 27 6 28 28 28 28 27 27 26 25 26 7 24 24 22 22 21 19 19 19 15 8 28 29 28 28 28 29 28 27 28 Mean 26.8 27.0 26.6 27.1 26.8 26.5 26.1 25.6 25.6 SEM 0.5 0.6 0.7 0.8 0.8 1.1 1.1 1.0 1.6 5 OBI-888 10 mg/kg × 4 1 27 28 27 28 27 NA NA NA NA IV 2 29 30 30 30 30 NA NA NA NA (Once weekly) 3 26 26 26 27 28 NA NA NA NA 4 27 28 27 28 28 NA NA NA NA 5 25 26 26 26 26 NA NA NA NA 6 26 26 27 27 27 NA NA NA NA 7 26 26 26 27 26 NA NA NA NA 8 26 26 26 26 26 NA NA NA NA Mean 26.5 27.0 26.9 27.4 27.3 — — — — SEM 0.4 0.5 0.5 0.5 0.5 — — — — 6 Anti-CD30 ADC 3 mg/kg × 4 1 25 27 27 27 27 27 27 27 27 (OBI-910) IV 2 26 28 27 28 28 28 29 29 29 (Once weekly) 3 26 27 27 28 27 27 27 27 27 4 27 28 29 29 30 29 30 29 31 5 24 25 25 25 25 24 26 26 26 Mean 25.6 27.0 27.0 27.4 27.4 27.0 27.8 27.6 28.0 SEM 0.5 0.5 0.6 0.7 0.8 0.8 0.7 0.6 0.9 7 MMAE + 0.191 mg/kg × 4 1 27 27 27 28 28 28 28 28 29 OBI-888 IP 2 25 25 25 26 25 24 24 24 25 (Once weekly) + 3 24 25 26 26 25 25 25 24 24 10 mg/kg × 4 4 24 25 24 25 23 24 24 24 25 IV 5 30 30 30 31 30 31 32 31 32 (Once weekly) 6 died died died died died died died died died 7 26 26 26 26 26 26 26 26 26 8 26 27 27 27 26 27 27 27 26 Mean 26.0 26.4 26.4 27.0 26.1 26.4 26.6 26.3 26.7 SEM 0.8 0.7 0.7 0.8 0.9 0.9 1.1 1.0 1.1 8 MMAE 0.191 mg/kg × 4 1 27 27 27 27 26 27 28 28 27 IP 2 24 25 24 25 25 24 25 25 25 (Once weekly) 3 27 28 28 28 27 28 28 28 28 4 27 26 27 28 27 28 28 28 29 5 27 28 27 28 27 28 28 27 28 6 died died died died died died died died died 7 26 27 27 27 26 24 25 24 23 8 27 27 27 28 27 27 29 28 28 Mean 26.4 26.9 26.7 27.3 26.4 26.6 27.3 26.9 26.9 SEM 0.4 0.4 0.5 0.4 0.3 0.7 0.6 0.6 0.8

TABLE 8-4 Body Weight, Xenograft, Gastric, NCI-N87 in Female nu/nu Mice (Day 88-Day 100) Dose (mg/kg) Body Weight (g) Gr. Treatment (Route) No. Day88 Day91 Day95 Day98 Day100 1 Vehicle 10 mL/kg × 4 1 NA NA NA NA NA (PBS, pH 7.4) + (Once weekly) 2 NA NA NA NA NA Vehicle IP + IV 3 NA NA NA NA NA (25 mM (Once weekly) 4 NA NA NA NA NA Sodium Citrate, + 5 NA NA NA NA NA 100 mM NaCl, 6 NA NA NA NA NA pH 6.5) 7 NA NA NA NA NA 8 NA NA NA NA NA Mean — — — — — SEM — — — — — 2 ADC (OBI-999) 1 mg/kg × 4 1 27 26 27 27 28 IV 2 29 28 28 29 29 (Once weekly) 3 29 29 29 29 30 4 died died died died died 5 29 29 30 29 29 6 30 29 30 29 30 7 26 26 26 26 27 8 30 29 30 30 31 Mean 28.6 28.0 28.6 28.4 29.1 SEM 0.6 0.5 0.6 0.5 0.5 3 ADC (OBI-999) 3 mg/kg × 4 1 29 29 28 29 29 IV 2 31 31 31 31 31 (Once weekly) 3 28 28 28 28 28 4 27 27 27 27 28 5 27 28 28 27 29 6 27 27 28 27 28 7 died died died died died 8 26 26 27 26 26 Mean 27.9 28.0 28.1 27.9 28.4 SEM 0.6 0.6 0.5 0.6 0.6 4 ADC (OBI-999) 10 mg/kg × 4 1 28 28 28 29 29 IV 2 27 28 28 28 29 (Once weekly) 3 26 25 26 25 25 4 28 27 27 27 28 5 26 25 25 23 23 6 26 26 28 29 30 7 NA NA NA NA NA 8 27 27 27 26 26 Mean 26.9 26.6 27.0 26.7 27.1 SEM 0.3 0.5 0.4 0.8 1.0 5 OBI-888 10 mg/kg × 4 1 NA NA NA NA NA IV 2 NA NA NA NA NA (Once weekly) 3 NA NA NA NA NA 4 NA NA NA NA NA 5 NA NA NA NA NA 6 NA NA NA NA NA 7 NA NA NA NA NA 8 NA NA NA NA NA Mean — — — — — SEM — — — — — 6 Anti-CD30 ADC 3 mg/kg x 4 1 27 27 27 27 28 (OBI-910) IV 2 29 29 30 30 30 (Once weekly) 3 26 25 25 25 25 4 31 31 31 30 32 5 26 25 25 25 25 Mean 27.8 27.4 27.6 27.4 28.0 SEM 1.0 1.2 1.2 1.1 1.4 7 MMAE + 0.191 mg/kg × 4 1 29 28 29 29 30 OBI-888 IP 2 26 26 26 25 25 (Once weekly) + 3 24 23 22 21 21 10 mg/kg × 4 4 26 25 25 26 25 IV 5 32 32 33 32 33 (Once weekly) 6 died died died died died 7 25 25 25 24 24 8 27 25 26 25 25 Mean 27.0 26.3 26.6 26.0 26.1 SEM 1.0 1.1 1.3 1.3 1.5 8 MMAE 0.191 mg/kg × 4 1 28 28 28 29 28 IP 2 25 26 26 27 27 (Once weekly) 3 29 29 29 29 30 4 29 29 29 29 29 5 28 28 29 29 30 6 died died died died died 7 23 23 22 22 22 8 28 28 29 28 29 Mean 27.1 27.3 27.4 27.6 27.9 SEM 0.9 0.8 1.0 1.0 1.1

FIG. 17 showed the tumor growth curves in NCI-H526 implanted female nude (nu/nu) mice. Intravenous administration of ADC (OBI-999) at 1 mg/kg, exhibited robust anti-tumor activity over the course of the study compared to the vehicle control group. Significant anti-tumor activity (T/C value ≤42%) was achieved starting on Day 15 and continuing through to Day 53 with a maximum percent TGI of 83% on Day 53. Intravenous administration of ADC (OBI-999) at 3 mg/kg, exhibited robust anti-tumor activity over the course of the study compared to the vehicle control group. Significant anti-tumor activity (T/C value ≤42%) was achieved starting on Day 11 and continuing through to Day 53 with a maximum percent TGI of 97% on Day 53. Intravenous administration of ADC (OBI-999) at 10 mg/kg, exhibited robust anti-tumor activity over the course of the study compared to the vehicle control group. Significant anti-tumor activity (T/C value ≤42%) was achieved starting on Day 11 and continuing through to Day 53 with a maximum percent TGI of 97% on Day 53.

Weekly intravenous (IV) administration of OBI-888 at 10 mg/kg, exhibited modest anti-tumor activity over the course of the study compared to the vehicle control group (FIG. 17).

Weekly intravenous (IV) administration of test substance, Anti-CD30 ADC (OBI-910) at 10 mg/kg, exhibited robust anti-tumor activity over the course of the study compared to the vehicle control group. Significant anti-tumor activity (T/C value ≤42%) was achieved starting on Day 11 and continuing through to Day 53 with a maximum percent TGI of 75% on Day 53 (FIG. 17).

Weekly intraperitoneal (IP) administration of standard agent, MMAE at 0.191 mg/kg, exhibited moderate anti-tumor activity over the course of the study compared to the vehicle control group with a maximum percent TGI of 53% on Day 53 (FIG. 17).

Combination therapy of test substance OBI-888 at 10 mg/kg with standard agent MMAE at 0.191 mg/kg was associated with significant inhibition of tumor growth over the course of the study compared to the vehicle control group. Significant anti-tumor activity (T/C value ≤42%) was achieved starting on Day 11 and continuing through to Day 53 with a maximum percent TGI of 62% on Day 53 (FIG. 17).

FIG. 18 showed the body weight changes in NCI-H526 implanted female nude (nu/nu) mice. All test substances were well-tolerated and not associated with any significant body weight loss over the course of the study.

Example 5: Measurement of the Anti-Tumor Activity of the Exemplary Antibody in Nude Mice (Lung Cancer)

In a xenograft tumor model of human small cell lung cancer, viable NCI-H526 stage E carcinoma; variant small cell lung cancer cells (ATCC CRL-5811), were subcutaneously (SC) implanted (1×10⁶ cells with matrigel (1:0.8) in 0.2 mL/mouse) into the right flank of female nu/nu mice. Tumor implanted mice were divided into five treatment groups, each group containing eight animals, and test agent administrations were initiated one day after cell implantation (denoted as Day 1).

5.1 Test Substances and Dosing Pattern

Test substances ADC (OBI-999), OBI-888, and corresponding vehicle were formulated by diluting stock with a 25 mM sodium citrate, 100 mM NaCl buffer (pH 6.5) and administered intravenously (IV) once weekly for four weeks. Standard agent, MMAE antibody at 0.191 mg/kg, and corresponding vehicle (PBS pH 7.4) were administered intraperitoneally (IP) once weekly for four weeks. One treatment group received combination therapy of test substance, OBI-888 at 10 mg/kg, with MMAE at 0.191 mg/kg.

TABLE 9 Study Design for Anti-Tumor Activity of the exemplary antibody in Nude Mice (Lung cancer) Mice^(c, d) Dosage (nu/nu) Group Test Compound Route mL/kg mg/kg (female) 1 Vehicle^(a) + IP + IV 10 N/A 8 Vehicle^(b) 2 ADC (OBI-999)^(b) IV 10 10 8 3 OBI-888^(b) IV 10 10 8 4 MMAE^(a) + IP + IV 10 0.191 + 10 8 OBI-888^(b) 5 MMAE^(a) IP 10 0.191 8 ^(a)PBS, pH 7.4 (high concentration of MMAE will be stored in 100% DMSO and then is diluted with PBS, pH 7.4) ^(b)25 mM Sodium Citrate + 100 mM NaCl, pH 6.5 ^(c)Vehicle and test substances are administered once weekly for four weeks starting one day after tumor cell implantation (denoted as Day 1). ^(d)NCI-H526 at 1 × 10⁶ cells/mouse with matrigel (1:0.8) in 200 μL are injected subcutaneously into right flank of female nu/nu mice. Tumor size/body weight monitoring: twice weekly till Day 70 or the study is terminated when mean tumor volume in the vehicle control group reaches 2000 mm³. Pictures are required to be taken at sacrifice.

5.2 Cell Line

The NCI-H526 tumor cell line was purchased from American Type Culture Collection (ATCC CRL-5811, variant small cell lung carcinoma) and cultured in Eurofins Panlabs Taiwan, Ltd. The cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37° C. in 5% CO₂ incubator and implanted subcutaneously in the right flank of each mouse. 5.3 Animals

Female nu/nu nude, aged 6-7 weeks, were obtained from BioLasco Taiwan (under Charles River Laboratories Licensee) and used. The animals were housed in individually ventilated cages (IVC, 36 Mini Isolator system). The allocation for 5 animals was 27×20×14 in cm. All animals were maintained in a hygienic environment under controlled temperature (20-24° C.) and humidity (30-70%) with 12-hour light/dark cycle. Free access to standard lab diet [MFG (Oriental Yeast Co., Ltd., Japan)] and autoclaved tap water were granted. All aspects of this work including housing, experimentation, and animal disposal were performed in general accordance with the “Guide for the Care and Use of Laboratory Animals: Eighth Edition” (National Academies Press, Washington, D.C., 2011) in our AAALAC-accredited laboratory animal facility. In addition, the animal care and use protocol was reviewed and approved by the IACUC at Eurofins Panlabs Taiwan, Ltd.

5.4 Chemicals

Fetal bovine serum (Hyclone, USA), RPMI-1640 medium (ThermoFisher, USA) and Matrigel (Corning, USA) were used in this experiment.

5.5 Equipment

Calipers (Mitutoyo, Japan), Centrifuge 5810R (Eppendorf, Germany), CO₂ Incubator (Forma Scientific Inc., USA), Hematocytometer (Hausser Scientific Horsham, USA), Individually ventilated cages racks (36 Mini Isolator system, Tecniplast, Italy), Inverted microscope CK-40 (Olympus, Japan), System microscope E-400 (Nikon, Japan) and Vertical laminar flow (Tsao-Hsin, Taiwan).

5.6 Methods

The tumor volumes, body weights, mortality, and signs of overt toxicity were monitored and recorded twice weekly for 45 days. Tumor growth inhibition was calculated as T/C (treatment/control)×100%. A T/C value ≤42% compared to that of the vehicle control group was considered significant anti-tumor activity. Two-way ANOVA followed by Bonferroni test was used to ascertain the statistically significant significance of groups compared to respective vehicle control (*p<0.05).

5.7 Results

TABLE 10-1 Tumor volume, Xenograft, Lung, NCI-H526 in Female nu/nu Mice (Day 1-Day 25) Dose (mg/kg) Tumor Volume (mm³) Gr. Treatment (Route) No. Day1 Day4 Day8 Day11 Day15 Day18 Day22 Day25 1 Vehicle 10 mL/kg × 4 1 66 61 92 104 159 536 942 1548 (PBS, pH 7.4) + (Once weekly) 2 86 91 86 111 101 157 190 428 Vehicle IP + IV 3 71 76 85 99 157 368 949 1578 (25 mM 4 89 103 137 164 180 401 965 1383 Sodium Citrate, + 5 80 80 80 172 221 474 757 1303 100 mM NaCl, 6 70 73 69 123 189 356 615 920 pH 6.5) 7 90 89 99 121 203 490 647 787 8 65 82 72 133 183 449 760 1004 Mean 77 82 90 128 174 404 728 1119 SEM 4 4 8 9 13 41 91 143 2 ADC (OBI-999) 10 mg/kg × 4 1 66 31 92 104 159 536 942 1548 IV 2 86 91 86 111 101 157 190 428 (Once weekly) 3 71 76 85 99 157 368 949 1578 4 89 103 137 164 180 401 965 1383 5 80 80 80 172 221 474 757 1303 6 70 73 69 123 189 356 615 920 7 90 89 99 121 203 490 647 787 8 65 82 72 133 183 449 760 1004 Mean 77 82 90 128 174 404 728 1119 SEM 5 9 4 4 5 11 33 46 % TGI N/A −24 −8 34 49 76 83 85 % T/C 99 124 108 66 51 24^(#) 17^(#) 15^(#) 3 OBI-888 10 mg/kg × 4 1 66 101 94 108 171 216 551 981 IV 2 86 87 81 99 113 183 504 725 (Once weekly) 3 80 121 81 91 136 201 415 681 4 66 97 104 127 135 222 511 913 5 86 93 98 96 166 170 483 756 6 86 86 81 93 62 76 113 289 7 94 94 79 79 89 77 87 99 8 71 99 69 89 82 99 93 83 Mean 79 97 86 98 119 156 345 566* SEM 4 4 4 5 14 22 74 126 % TGI NA −18 4 23 32 61 53 49 % T/C 103 118 96 77 68 39^(#) 47 51 4 MMAE + 0.191 mg/kg × 4 1 82 86 93 119 112 179 323 511 OBI-888 IP 2 63 82 83 69 69 66 68 118 (Once weekly) + 3 68 108 94 83 61 95 148 346 10 mg/kg × 4 4 87 80 79 148 142 181 525 938 IV 5 97 81 93 96 121 141 402 590 (Once weekly) 6 101 111 88 93 98 98 119 171 7 93 99 89 99 115 137 388 540 8 87 94 94 88 115 122 333 507 Mean 85 93 89 99 104 127 288* 465* SEM 5 4 2 9 10 14 57 92 % TGI NA −13 1 23 40 69 60 58 % T/C 110 113 99 77 60 31^(#) 40^(#) 42^(#) 5 MMAE 0.191 mg/kg × 4 1 75 80 94 74 82 80 74 70 IP 2 108 127 74 86 133 199 618 1163 (Once weekly) 3 81 101 94 89 137 246 530 1095 4 83 88 83 104 101 169 337 483 5 99 115 70 121 144 187 317 525 6 60 85 82 67 89 101 152 249 7 68 80 121 77 172 281 621 1078 8 91 108 74 79 123 202 401 535 Mean 83 98 87 87 123 183 381 650* SEM 6 6 6 6 11 24 72 146 % TGI NA −20 3 32 29 55 48 42 % T/C 108 120 97 68 71 45 52 58

TABLE 10-2 Tumor volume, Xenograft, Lung, NCI-H526 in Female nu/nu Mice (Day 29-Day 45) Dose (mg/kg) Tumor Volume (mm³) Gr. Treatment (Route) No. Day29 Day31 Day36 Day39 Day43 Day45 1 Vehicle 10 mL/kg × 4 1 1968 2452 NA NA NA NA (PBS, pH 7.4) + (Once weekly) 2 968 1251 NA NA NA NA Vehicle IP + IV 3 2579 3369 NA NA NA NA (25 mM 4 2218 2803 NA NA NA NA Sodium Citrate, + 5 2342 2329 NA NA NA NA 100 mM NaCl, 6 1594 1794 NA NA NA NA pH 6.5) 7 1561 2022 NA NA NA NA 8 1942 2363 NA NA NA NA Mean 1897 2298 — — — — SEM 181 226 — — — — 2 ADC (OBI-999) 10 mg/kg × 4 1 517 717 922 died died died IV 2 207 289 612 615 953 1095 (Once weekly) 3 811 983 1886 2403 3693 4092 4 99 79 0 0 0 0 5 507 644 1349 1798 2982 3948 6 231 333 789 1094 1727 2190 7 150 265 461 702 1109 1369 8 80 111 144 218 318 395 Mean 325* 428* 770 976 1540 1870 SEM 92 113 220 305 482 575 % TGI 83 81 — — — — % TIC 17^(#) 19^(#) — — — — 3 OBI-888 10 mg/kg × 4 1 1640 1837 3370 3941 NA NA IV 2 1227 1519 2820 3803 NA NA (Once weekly) 3 931 1246 2045 2174 NA NA 4 1318 1714 2856 3617 NA NA 5 1176 1539 1998 2177 NA NA 6 500 550 1159 1802 NA NA 7 120 214 322 410 NA NA 8 77 101 70 63 NA NA Mean 874* 1090* 1830 2248 — — SEM 205 246 429 526 — — % TGI 54 53 — — — — % T/C 46 47 — — — — 4 MMAE + 0.191 mg/kg × 4 1 747 866 1514 2347 NA NA OBI-888 IP 2 184 321 877 1485 NA NA (Once weekly) + 3 632 887 1897 2822 NA NA 10 mg/kg × 4 4 1654 2176 3764 5272 NA NA IV 5 1150 1437 2654 3181 NA NA (Once weekly) 6 389 636 982 1333 NA NA 7 1046 1204 2056 3536 NA NA 8 1034 1367 2251 3438 NA NA Mean 855* 1112* 1999 2927 — — SEM 165 202 331 446 — — % TGI 55 52 — — — — % T/C 45 48 — — — — 5 MMAE 0.191 mg/kg × 4 1 90 173 126 56 NA NA IP 2 1756 1901 3047 4380 NA NA (Once weekly) 3 1410 1682 2480 2713 NA NA 4 853 1172 2090 2836 NA NA 5 522 657 759 841 NA NA 6 431 550 1032 1304 NA NA 7 1313 1595 2538 3040 NA NA 8 845 1044 1318 1339 NA NA Mean 903* 1097* 1674 2064 — — SEM 198 215 359 499 — — % TGI 52 52 — — — — % T/C 48 48 — — — —

TABLE 11-1 Body weight, Xenograft, Lung, NCI-H526 in Female nu/nu Mice (Day 1-Day 25) Dose (mg/kg) Body Weight (g) Gr. Treatment (Route) No. Day1 Day4 Day8 Day11 Day15 Day18 Day22 Day25 1 Vehicle 10 mL/kg × 4 1 23 24 24 23 24 25 26 27 (PBS, pH 7.4) + (Once weekly) 2 23 25 26 25 26 25 25 26 Vehicle IP + IV 3 23 24 24 25 25 26 25 26 (25 mM 4 25 25 25 24 24 25 26 27 Sodium Citrate, + 5 24 24 24 24 24 26 26 27 100 mM NaCl, 6 25 26 26 25 26 26 27 27 pH 6.5) 7 24 25 26 25 24 26 26 26 8 22 23 22 22 23 24 24 25 Mean 23.6 24.5 24.6 24.1 24.5 25.4 25.6 26.4 SEM 0.4 0.3 0.5 0.4 0.4 0.3 0.3 0.3 1 24 25 25 25 25 26 26 26 2 24 24 25 25 25 25 25 25 3 26 26 28 28 27 28 28 28 4 24 25 24 24 25 27 27 27 2 ADC (OBI-999) 10 mg/kg × 4 5 25 26 28 28 28 28 28 29 IV 6 24 25 26 26 25 26 25 26 (Once weekly) 7 23 24 25 24 24 24 24 24 8 24 24 25 25 25 25 25 26 Mean 24.3 24.9 25.8 25.6 25.5 26.1 26.0 26.4 SEM 0.3 0.3 0.5 0.6 0.5 0.5 0.5 0.6 1 25 25 27 27 27 28 28 29 2 24 24 25 25 25 25 26 27 3 24 23 24 23 24 25 24 25 4 25 25 27 28 28 29 29 30 3 OBI-888 10 mg/kg × 4 5 24 24 24 25 25 26 26 27 IV 6 26 27 28 28 28 29 29 30 (Once weekly) 7 25 26 26 26 27 27 27 27 8 24 24 25 25 26 25 26 26 Mean 24.6 24.8 25.8 25.9 26.3 26.8 26.9 27.6 SEM 0.3 0.5 0.5 0.6 0.5 0.6 0.6 0.7 4 MMAE + 0.191 mg/kg × 4 1 25 25 26 25 26 26 27 27 OBI-888 IP 2 24 24 27 27 28 29 29 28 (Once weekly) + 3 24 24 26 25 26 27 26 26 10 mg/kg × 4 4 25 22 24 25 26 26 26 27 IV 5 24 24 26 27 28 28 28 28 (Once weekly) 6 25 26 27 27 28 28 28 29 7 25 26 27 27 28 27 28 29 8 21 21 23 24 25 25 25 25 Mean 24.1 24.0 25.8 25.9 26.9 27.0 27.1 27.4 SEM 0.5 0.6 0.5 0.4 0.4 0.5 0.5 0.5 5 MMAE 0.191 mg/kg × 4 1 24 22 24 24 25 25 24 26 IP 2 25 25 26 28 28 28 28 30 (Once weekly) 3 26 27 28 28 28 28 28 29 4 24 21 21 23 24 24 25 26 5 24 23 25 24 25 25 25 25 6 23 23 23 23 23 24 24 25 7 23 24 24 24 24 24 25 25 8 22 24 25 25 26 25 25 26 Mean 23.9 23.6 24.5 24.9 25.4 25.4 25.5 26.5 SEM 0.4 0.7 0.7 0.7 0.7 0.6 0.6 0.7

TABLE 11-2 Body weight, Xenograft, Lung, NCI-H526 in Female nu/nu Mice (Day 29-Day 45) Dose (mg/kg) Body Weight (g) Gr. Treatment (Route) No. Day29 Day31 Day36 Day39 Day43 Day45 1 Vehicle 10 mL/kg × 4 1 28 29 NA NA NA NA (PBS, pH 7.4) + (Once weekly) 2 27 28 NA NA NA NA Vehicle IP + IV 3 28 30 NA NA NA NA (25 mM 4 30 30 NA NA NA NA Sodium Citrate, + 5 29 30 NA NA NA NA 100 mM NaCl, 6 29 30 NA NA NA NA pH 6.5) 7 28 29 NA NA NA NA 8 26 28 NA NA NA NA Mean 28.1 29.3 — — — — SEM 0.4 0.3 — — — — 2 ADC (OBI-999) 10 mg/kg × 4 1 27 25 24 died died died IV 2 26 26 27 27 27 28 (Once weekly) 3 29 29 31 32 34 36 4 28 27 28 29 28 29 5 32 31 32 34 33 35 6 27 27 28 29 30 31 7 25 25 26 27 26 28 8 26 26 27 28 27 28 Mean 27.5 27.0 27.9 29.4 29.3 30.7 SEM 0.8 0.7 0.9 0.9 1.1 1.2 3 OBI-888 10 mg/kg × 4 1 32 31 35 36 NA NA IV 2 28 28 30 30 NA NA (Once weekly) 3 26 25 27 28 NA NA 4 32 31 33 36 NA NA 5 28 27 27 28 NA NA 6 31 31 32 34 NA NA 7 27 27 29 29 NA NA 8 27 27 27 27 NA NA Mean 28.9 28.4 30.0 31.0 — — SEM 0.9 0.8 1.1 1.3 — — 4 MMAE + 0.191 mg/kg × 4 1 29 29 31 33 NA NA OBI-888 IP 2 30 30 31 32 NA NA (Once weekly) + 3 28 28 31 32 NA NA 10 mg/kg × 4 4 30 30 33 35 NA NA IV 5 30 30 32 34 NA NA (Once weekly) 6 30 30 30 34 NA NA 7 32 32 33 35 NA NA 8 27 27 29 33 NA NA Mean 29.5 29.5 31.3 33.5 — — SEM 0.5 0.5 0.5 0.4 — — 5 MMAE 0.191 mg/kg × 4 1 25 25 26 27 NA NA IP 2 32 32 33 38 NA NA (Once weekly) 3 31 32 33 34 NA NA 4 26 26 28 30 NA NA 5 27 27 27 27 NA NA 6 26 26 26 27 NA NA 7 27 27 29 30 NA NA 8 27 27 29 29 NA NA Mean 27.6 27.8 28.9 30.3 — — SEM 0.9 1.0 1.0 1.4 — —

FIG. 27 showed the tumor growth curves in NCI-H526 implanted female nude (nu/nu) mice. Intravenous administration of ADC (OBI-999) at 10 mg/kg once weekly for four weeks was associated with significant anti-tumor activity (T/C value ≤42%) starting on Day 15 and continued through to Day 31 with a maximum percent TGI of 85% on Day 25.

Weekly intravenous (IV) administration of test substance, OBI-888 at 10 mg/kg, exhibited moderate anti-tumor activity over the course of the study compared to the vehicle control group; however, significant anti-tumor activity (T/C value ≤42%) was achieved on Day 18 of the study with a maximum percent TGI of 61% on Day 18.

Weekly intraperitoneal (IP) administration of standard agent, MMAE at 0.191 mg/kg, exhibited moderate anti-tumor activity over the course of the study compared to the vehicle control group with a maximum percent TGI of 55% on Day 18.

Combination therapy of test substance OBI-888 at 10 mg/kg with standard agent MMAE at 0.191 mg/kg was associated with moderate inhibition of tumor growth over the course of the study compared to the vehicle control group; however, significant anti-tumor activity (T/C value ≤42%) was achieved on Day 18, Day 22, and Day 25 with a maximum percent TGI of 69% on Day 18.

FIG. 28 showed the body weight changes in NCI-H526 implanted female nude (nu/nu) mice. All test substances were well-tolerated and not associated with any significant body weight loss over the course of the study.

Example 6: Measurement of the Anti-Tumor Activity of the Exemplary Antibody in Nude Mice (Pancreatic Cancer)

The objective of this study was to evaluate the in vivo anti-tumor efficacy of OBI-888, ADC (OBI-999), MMAE and OBI-888 combined with MMAE in HPAC human pancreatic cancer xenograft model in male BALB/c nude mice.

6.1 Test Substances and Dosing Pattern

Test substances ADC (OBI-999), OBI-888, and corresponding vehicle were formulated by diluting stock with a 25 mM sodium citrate, 100 mM NaCl buffer (pH 6.5) and administered intravenously (IV) once weekly for four weeks. Standard agent, MMAE antibody at 0.191 mg/kg, and corresponding vehicle (PBS pH 7.4) were administered intraperitoneally (IP) once weekly for four weeks. One treatment group received combination therapy of test substance, OBI-888 at 10 mg/kg, with MMAE at 0.191 mg/kg.

TABLE 12 Study Design for Anti-Tumor Activity of the exemplary antibody in Nude Mice (Pancreatic cancer) Mice^(c, d) Dosage (nu/nu) Group Test Compound Route mL/kg mg/kg (male) 1 Vehicle^(a) + IP + IV 10 N/A 8 Vehicle^(b) 2 ADC (OBI-999)^(b) IV 10 10 8 3 OBI-888^(b) IV 10 10 8 4 MMAE^(a) + IP + IV 10 0.191^(a) + 10^(b) 8 OBI-888^(b) 5 MMAE^(a) IP 10 0.191 8 ^(a)PBS, pH 7.4 (high concentration of MMAE will be stored in 100% DMSO and then is diluted with PBS, pH 7.4) ^(b)25 mM Sodium Citrate + 100 mM NaCl, , pH 6.5 ^(c)Vehicle and test substances are administered once weekly for four weeks starting one day after tumor cell implantation (denoted as Day 1). ^(d)Each mouse was inoculated subcutaneously with HPAC tumor cells (3 × 10⁶) in 0.2 mL of PBS for tumor development. Treatments were started on day 6 after tumor inoculation when the average tumor size reached 85 mm³.

6.2 Cell Line

The HPAC tumor cells (ATCC CRL-2119) were maintained in vitro as a monolayer culture in 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium containing 1.2 g/L sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES and 0.5 mM sodium pyruvate supplemented with 0.002 mg/mL insulin, 0.005 mg/mL transferrin, 40 ng/mL hydrocortisone, 10 ng/mL epidermal growth factor and 5% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin at 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells were routinely subcultured twice weekly by trypsin-EDTA treatment. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

6.3 Animals

Male nu/nu nude, aged 6-8 weeks, were obtained from Shanghai Lingchang and used. The mice were kept in individual ventilation cages at constant temperature and humidity with four animals in each cage (temperature: 20-26° C. and humidity: 40-70%). The cages were made of polycarbonate and the size was 300 mm×200 mm×180 mm. The bedding material was corn cob, which was changed twice per week. Animals had free access to irradiation sterilized dry granule food and drinking water during the entire study period. The identification labels for each cage contained the following information: number of animals, sex, strain, date received, treatment, study number, group number and the starting date of the treatment.

6.4 Methods

The endpoint was to determine the anti-tumor effects of testing compounds. Tumor size was measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm³ using the formula: V=0.5 a×b² where a and b are the long and short diameters of the tumor, respectively. The tumor size was then used for calculation of T/C values. The T/C value (in percent) is an indication of antitumor effectiveness; T and C are the mean volumes of the treated and control groups, respectively, on a given day. TGI was calculated for each group using the formula: TGI (%)=[1−(Ti−T0)/(Vi−V0)]×100; Ti is the average tumor volume of a treatment group on a given day, T0 is the average tumor volume of the treatment group on day 0, Vi is the average tumor volume of the vehicle control group on the same day with Ti, and V0 is the average tumor volume of the vehicle group on day 0.

Summary statistics, including mean and the standard error of the mean (SEM), are provided for the tumor volume of each group at each time point. Statistical analysis of difference in the tumor volume among the groups were conducted on the data obtained at the best therapeutic time point after the final dose (the 37^(th) day after grouping). A one-way ANOVA was performed to compare the tumor volume among groups, and when a significant F-statistics (a ratio of treatment variance to the error variance) was obtained, comparisons between groups were carried out with Games-Howell test, otherwise they were carried out with Dunnett (2 sided) test. The potential synergistic effect between OBI-888 and MMAE was analyzed by two-way ANOVA. All data were analyzed using SPSS 17.0. p<0.05 was considered to be statistically significant.

6.5 Results

TABLE 13 Tumor volume, pancreas, HPAC in nu/nu Mice Tumor Volume (mm³) Treatment No. 0^(a) 3 7 10 14 17 21 24 28 31 35 37 Group-1 1 87 432 503 628 1060 1179 1259 1508 2143 2950 4426 4239 Vehicle A + B 2 93 104 203 251 468 654 929 1287 1471 1589 1560 1792 IP + IV 3 134 176 208 284 485 636 842 939 1263 1431 1465 1881 10 μL/g + 4 80 124 161 252 341 735 979 1024 1729 1627 1692 1866 10 μL/g 5 61 204 253 378 492 595 896 876 1079 1292 1289 1953 QW × 4 6 111 161 203 343 501 637 670 725 1078 1549 1629 2178 7 54 77 141 188 334 388 513 567 818 1033 1161 1450 8 59 71 135 186 320 428 653 762 994 1227 1359 1998 Mean 85 169 226 314 500 656 843 961 1322 1587 1823 2170 SEM 10 41 42 51 85 85 82 109 155 207 377 305 Group-2 1 59 75 104 135 53 26 4 1 0 0 0 0 ADC (OBI-999) 2 80 115 124 116 43 4 1 0 0 0 0 0 IV 3 100 74 75 61 0 0 0 0 0 0 0 0 10 mg/kg 4 56 94 115 91 0 0 0 0 0 0 0 0 QW × 4 5 101 148 196 178 102 76 15 14 12 4 1 0 6 122 149 264 180 134 65 52 22 18 20 4 12 7 72 76 101 87 42 16 4 1 1 0 0 0 8 89 154 175 79 0 0 0 0 0 0 0 0 Mean 85 111 144 116 47 23 9 5 4 3 1 1 SEM 8 13 22 16 18 11 6 3 2 2 0 1 Group-3 1 140 155 170 251 384 404 781 874 1471 1952 2063 2073 OBI-888 2 52 61 154 209 412 523 695 952 999 1489 1535 1839 IV 3 84 128 169 331 481 571 772 908 1480 1722 2696 2620 10 mg/kg 4 90 100 140 296 323 442 671 992 1590 1915 2275 2269 QW × x 4 5 109 98 129 252 458 615 727 870 1200 1627 1836 1838 6 58 71 116 214 255 303 645 635 1134 1175 1485 1791 7 75 98 219 367 529 641 724 857 1150 1422 1584 1852 8 69 151 164 288 610 706 930 1132 1663 1876 2046 2074 Mean 85 108 158 276 432 526 743 902 1336 1647 1940 2044 SEM 10 12 11 20 40 48 31 50 86 96 148 101 Group-4 1 128 166 189 302 520 578 656 844 971 1370 1440 1640 MMAE + 2 118 100 108 154 286 366 453 717 863 904 1332 1577 OBI-888 3 45 79 Died IP + IV 4 88 93 143 243 371 824 898 1134 1606 1632 1830 2226 0.191 mg/kg + 5 71 75 161 200 279 451 486 693 840 1186 1218 1227 10 mg/kg 6 79 112 121 220 288 414 483 577 985 1063 1192 1638 QW × 4 7 91 111 244 274 561 653 735 1292 1507 2073 2400 2523 8 57 53 73 89 110 189 293 359 554 844 940 931 Mean 85 99 148 212 345 496 572 802 1047 1296 1479 1680 SEM 10 12 21 27 59 79 77 121 143 165 185 206 Group-5 1 58 128 Died MMAE 2 53 76 104 Euthanized IP 3 132 148 Died 0.191 mg/kg 4 72 82 114 444 429 590 649 748 1080 1174 1650 1652 QW × 4 5 86 158 177 196 418 452 692 705 888 1340 1656 1963 6 116 128 144 219 418 510 581 822 913 1439 1496 1828 7 71 57 97 160 268 321 383 511 623 1030 1236 1196 8 91 137 167 207 390 448 451 571 785 989 1208 1580 Mean 85 114 134 245 385 464 551 672 858 1194 1449 1644 SEM 10 13 14 51 30 44 59 57 75 87 97 130

TABLE 14 Body weight, pancreas, HPAC in nu/nu Mice Body weight (g) Treatment No. 0^(a) 3 4 7 9 10 14 17 21 24 28 31 35 37 Group-1 1 24.8 24.7 24.4 25.3 25.5 25.6 26.6 26.8 27.2 27.2 27.4 26.9 27.9 28.5 Vehicle 2 21.9 22.0 22.2 22.7 23.0 22.9 23.5 23.6 23.8 23.9 24.5 23.7 24.4 24.8 A + B 3 24.7 24.3 24.5 25.3 25.5 25.5 26.6 26.5 26.6 26.8 27.1 26.4 28.2 28.3 IP + IV 4 21.0 21.4 21.8 22.5 22.5 22.7 23.2 23.3 24.2 24.3 25.1 24.7 25.2 26.2 10 μL/g+ 5 23.3 24.1 24.6 25.4 25.0 25.0 25.5 25.4 25.6 26.0 27.0 26.2 27.0 27.2 10 μL/g 6 21.4 22.3 22.4 23.0 23.0 23.4 23.5 23.2 23.2 23.6 23.5 23.9 23.5 24.3 QW × 4 7 22.8 23.0 23.5 24.2 24.2 24.1 24.4 24.2 24.9 25.0 25.4 25.6 26.8 27.2 8 24.4 24.8 25.0 25.7 26.9 25.7 26.3 22.2 26.6 27.0 27.4 27.5 28.3 28.5 Mean 23.0 23.3 23.5 24.2 24.4 24.4 25.0 24.4 25.3 25.5 25.9 25.6 26.4 26.9 SEM 0.5 0.5 0.4 0.5 0.6 0.4 0.5 0.6 0.5 0.5 0.5 0.5 0.6 0.6 Group-2 1 24.4 24.9 24.8 25.5 25.7 25.9 26.6 26.4 26.2 26.3 26.3 26.6 26.8 27.0 ADC 2 24.1 24.9 25.5 25.6 25.8 25.7 26.9 26.7 27.3 27.4 27.1 27.7 27.9 28.1 (OBI-999) 3 23.8 23.9 23.9 24.5 24.8 24.6 25.6 25.6 25.9 26.1 26.0 26.4 27.0 27.8 IV 4 23.2 24.6 24.4 24.9 25.3 25.1 25.1 24.6 25.1 25.3 25.3 25.3 24.3 24.9 10 mg/kg 5 24.5 25.1 25.3 25.9 25.8 25.8 26.8 26.5 27.4 27.4 27.4 27.5 27.6 28.9 QW × 4 6 24.8 24.4 25.1 25.8 26.2 26.3 27.4 26.7 26.3 26.3 26.9 26.9 27.3 27.9 7 22.8 23.0 23.1 24.0 23.8 23.8 24.3 24.0 24.5 24.6 24.6 24.7 24.5 25.6 8 24.6 24.3 24.4 25.4 25.4 25.3 26.1 26.3 26.4 26.1 26.4 26.4 27.1 27.6 Mean 24.0 24.4 24.6 25.2 25.3 25.3 26.1 25.9 26.1 26.2 26.3 26.4 26.6 27.2 SEM 0.3 0.2 0.3 0.2 0.3 0.3 0.4 0.4 0.3 0.3 0.3 0.4 0.5 0.5 Group-3 1 25.1 24.3 24.5 25.5 25.4 25.8 26.5 26.9 26.4 27.3 26.9 26.8 28.8 28.4 OBI-888 2 25.5 25.5 25.9 26.6 27.3 27.4 27.0 27.3 27.8 29.4 28.8 28.4 28.9 29.0 IV 3 24.9 24.4 24.9 25.5 26.2 26.5 27.0 27.6 27.7 27.9 28.5 27.8 29.1 29.1 10 mg/kg 4 25.0 25.5 26.1 27.1 27.0 27.1 24.5 27.6 27.3 26.8 27.9 27.5 28.5 27.9 QW × 4 5 24.3 23.7 24.0 24.1 24.8 25.0 26.0 26.5 26.3 25.0 26.2 26.1 27.1 27.1 6 23.9 24.5 24.7 25.2 25.3 25.1 25.6 25.6 25.7 26.7 26.9 26.2 26.8 26.9 7 24.4 24.6 24.7 25.0 25.4 25.5 25.9 26.2 26.4 26.5 27.2 26.8 27.5 27.7 8 23.0 23.5 23.7 23.9 24.5 24.9 25.1 24.9 24.6 24.7 25.3 25.4 25.9 25.3 Mean 24.5 24.5 24.8 25.4 25.7 25.9 25.9 26.6 26.5 26.8 27.2 26.9 27.8 27.7 SEM 0.3 0.3 0.3 0.4 0.4 0.3 0.3 0.3 0.4 0.5 0.4 0.3 0.4 0.4 Group-4 1 27.2 23.6 23.9 26.9 27.1 27.5 28.3 28.0 28.2 27.8 28.3 29.7 30.4 29.9 MMAE + 2 25.1 23.7 24.0 26.3 25.9 26.2 26.7 26.6 26.9 27.0 26.9 27.4 28.2 28.0 OBI-888 3 23.6 21.6 20.7 Died IP + IV 4 24.1 21.7 22.6 25.6 25.4 25.3 25.8 25.4 26.2 25.2 25.4 24.9 25.5 25.6 0.191 mg/kg + 5 23.9 22.6 23.6 24.3 24.0 24.3 25.1 24.6 24.5 24.7 24.3 23.8 25.0 25.1 10 mg/kg 6 25.2 23.7 25.0 27.5 27.7 27.9 28.8 26.9 28.3 28.4 28.3 28.4 29.7 30.0 QW × 4 7 24.3 22.3 21.2 23.7 23.9 24.2 25.4 26.0 26.1 26.7 26.3 26.8 28.1 28.3 8 23.3 23.7 24.1 24.6 25.2 25.6 25.2 26.0 26.1 26.7 26.3 26.8 27.5 27.6 Mean 24.6 22.8 23.1 25.5 25.6 25.8 26.5 26.2 26.6 26.6 26.5 26.8 27.8 27.8 SEM 0.4 0.3 0.5 0.5 0.5 0.5 0.6 0.4 0.5 0.5 0.6 0.7 0.8 0.7 Group-5 1 25.4 21.8 21.0 Died MMAE 2 24.3 22.4 21.3 18.8 Euthanized IP 3 24.8 21.5 20.5 Died 0.191 mg/kg 4 24.2 21.6 22.0 25.1 24.9 25.9 25.7 23.9 25.6 24.8 26.2 25.0 26.9 26.7 QW × 4 5 24.5 24.0 24.9 27.0 25.9 26.0 26.2 26.0 26.2 25.7 25.3 25.2 25.6 25.3 6 23.3 20.7 19.6 21.7 22.2 26.3 24.4 24.5 24.7 24.9 24.9 25.4 25.6 25.5 7 24.0 23.1 23.7 25.2 24.9 25.3 26.0 25.3 25.7 25.6 26.3 26.4 27.2 27.1 8 22.7 21.6 22.1 23.8 23.5 24.1 24.5 23.6 23.8 23.7 24.1 24.1 24.5 24.0 Mean 24.2 22.1 21.9 23.6 24.3 25.5 25.3 24.6 25.2 24.9 25.4 25.2 26.0 25.7 SEM 0.3 0.4 0.6 1.2 0.7 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.5 0.5

FIG. 34 showed the tumor growth curves in HPAC implanted nude (nu/nu) mice. Treatment with the test article ADC (OBI-999) at 10 mg/kg produced a significant antitumor activity starting on Day 14 and continued through to Day 37. Its mean tumor size was 1 mm³ (T/C=0.1%, TGI=104.0%, p<0.001). OBI-888 at 10 mg/kg as a single agent didn't produce significant antitumor activity. Its mean tumor size was 2,044 mm³ (T/C=94.2%, TGI=6.0%, p=0.967). MMAE at 0.191 mg/kg as a single agent or combined with OBI-888 at 10 mg/kg produced a minor antitumor activity with a mean tumor size of 1,644 mm³ (T/C=75.8%, TGI=25.2%, p=0.231) and 1,680 mm³ (T/C=77.4%, TGI=23.5%, p=0.213), respectively.

FIG. 35 showed the body weight changes in HPAC implanted nude (nu/nu) mice. All test substances were well-tolerated and not associated with any significant body weight loss over the course of the study.

Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of this invention. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used to practice this invention, the preferred compositions, methods, kits, and means for communicating information are described herein.

All references cited herein are incorporated herein by reference to the full extent allowed by law. The discussion of those references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference. 

What is claimed is:
 1. A method of treating cancer in a subject, the method comprising administering to the subject in need thereof an effective amount of an antibody-drug conjugate (ADC); wherein the ADC comprising a therapeutic agent and an antibody or an antigen-binding fragment that binds Globo H (Fucα1→2 Galβ1→3 GalNAcβ1→3 Galα1→4 Galβ1→4 Glc); wherein the therapeutic agent is covalently conjugated to the antibody or the antigen-binding fragment by a linker; and wherein the anti-Globo H antibody is OBI-888.
 2. The method of claim 1, wherein the cancer is Globo H expressing cancer.
 3. The method of claim 2, wherein the Globo H expressing cancer is selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testical cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer, prostate cancer and gastric cancer.
 4. The method of claim 3, wherein the Globo H expressing cancer is selected from the group consisting of breast cancer, pancreatic cancer, gastric cancer and lung cancer.
 5. The method of claim 1, wherein the subject is human.
 6. The method of claim 1, wherein the effective amount of the ADC is administered intravenously.
 7. The method of claim 1, wherein the method comprising administering to the subject in need thereof an effective amount of a combination of other ADCs targeting Globo series antigens.
 8. The method of claim 7, wherein the Globo series antigens is SSEA-4 (Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1), SSEA-3 (Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1) or Globo H.
 9. The method of claim 7, wherein the combination provides a synergistic effect in cancer treatment and enhanced therapeutic efficacy.
 10. A method of inducing or enhancing immune reaction in a subject in need thereof comprising: administering an immunogenically effective amount of the pharmaceutical composition and one or more of the following procedures selected from: administering the ADC of claim 1 two or more times; adjusting time interval and/or dosing amount regimen between two successive administrations; adjusting routes of administration and/or altering injection sites of administration; or combining other ADCs comprising Globo series antigens; wherein the composition comprising a mixture of ADC of claim 1 having the formula: Ab-(L-D)_(n)  (I); wherein one or more therapeutic drug moieties (D) are covalently linked by a linker (L) to an antibody (Ab); wherein the antibody is an anti-Globo H antibody; and wherein n is an integer from 1 to
 8. 11. The method of claim 10, wherein the Globo series antigens is SSEA-4 (Neu5Acα2→3Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1), SSEA-3 (Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glcβ1) or Globo H.
 12. The method of claim 10, wherein the injections can be altered and/or supplemented by the addition of immune response booster agents.
 13. The method of claim 10, wherein the subject is human.
 14. The method of claim 10, wherein the effective amount is from 0.01 μg to 250 mg.
 15. The method of claim 10, wherein the effective amount is from 0.001 μg/kg to 250 mg/kg.
 16. The method of claim 10, wherein the effective amount of the ADC is administered intravenously.
 17. The method of claim 10, wherein the effective amounts are in a co-formulation or separate formulations.
 18. A process of making the ADC of claim 1 in the manufacture of a medicament for the treatment of Globo H expressing cancer.
 19. The process of claim 18, wherein the Globo H expressing cancer is selected from the group of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testical cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer, prostate cancer and gastric cancer.
 20. The method of claim 1, wherein the ADC is administered in combination with an effective amount of an additional agent selected from the group consisting of an anticancer agent, an immunosuppressant agent, and an anti-infectious agent for the treatment of muscular disorders, ubiquitin-pathway-related genetic disorders, immune/inflammatory disorders, neurological disorders, and other ubiquitin pathway-related disorders.
 21. The method of claim 20, wherein the combination is administered via combined administration or separate administration.
 22. A kit comprising an immunogenically effective amount of the pharmaceutical composition of claim 10 for the treatment of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testical cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer, prostate cancer or gastric cancer. 